Use of self-assembling polypeptides as tissue adhesives

ABSTRACT

The present invention relates to a self-assembling polypeptide for use as tissue adhesive. The present invention also relates to the use of a self-assembling polypeptide as tissue adhesive. Further, the invention is directed to the use of a self-assembling polypeptide to glue one or more cosmetic compounds on skin, mucosa, and/or hair. Furthermore, the invention is directed to a self-assembling polypeptide for use in gluing one or more pharmaceutical compounds on tissue, skin, mucosa, and/or hair.

The present invention relates to a self-assembling polypeptide for useas tissue adhesive. The present invention also relates to the use of aself-assembling polypeptide as tissue adhesive. Further, the inventionis directed to the use of a self-assembling polypeptide to glue one ormore cosmetic compounds on skin, mucosa, and/or hair. Furthermore, theinvention is directed to a self-assembling polypeptide for use in gluingone or more pharmaceutical compounds on tissue, skin, mucosa, and/orhair.

BACKGROUND OF THE INVENTION

Tissue adhesives continue to evolve as an important technology for thephysician, particularly surgeon. Years ago there was little routine useof these substances; however, in the past years there have beensignificant advances. It is becoming increasingly important for thephysician, particularly surgeon, to be familiar with the indications andshortcomings of these compounds. Currently available tissue adhesivescan be categorized as either fibrin tissue adhesives or acrylate-basedtissue adhesives, e.g. cyanoacrylates. Although fibrin tissue adhesivesand acrylate-based tissue adhesives, e.g. cyanoacrylates, are oftendiscussed under the general topic of tissue adhesives, these twosubstances have different indications and mechanisms of action. Fibrintissue adhesives use naturally occurring substrates that are part ofnormal endogenous clotting mechanisms. In contrast, the adhesionachieved by acrylate-based tissue adhesives, e.g. cyanoacrylates, is aresult of synthetic compounds not naturally occurring in the human oranimal body. These two types of adhesives also have different clinicalindications. Fibrin tissue adhesives are typically applied below thedermis as a biologic hemostat or as a sealant for use with skin graftsand flaps. Acrylate-based tissue adhesives, e.g. cyanoacrylates, havebeen used most successfully at the level of the epidermis forsuperficial skin closure (see Toriumi D M, Raslan W F, Friedman M, etal. “Histotoxicity of cyanoacrylate tissue adhesives.”, Arch OtolaryngolHead Neck Surg 1990; 116: 546-50).

The mechanism of action of fibrin tissue adhesives is best understood byreviewing basic blood coagulation physiology. During the normal clottingprocess, thrombin cleaves the large molecular weight protein fibrinogeninto smaller fibrin subunits. These subunits then undergo bothend-to-end and side-to-side polymerization. Factor XIII (plasmaglutaminase), in the presence of calcium, enables the cross-linking ofthese polymerized subunits into a stable fibrin clot. Usually, fibrintissue adhesives are packaged as two separate components that when mixedon the injured or surgical field simulate the interaction of theseendogenous compounds and form the final fibrin clot. The first componentis composed of fibrinogen, factor XIII, and calcium chloride, while thesecond component is made up of thrombin and an antifibrinolytic agent.Fibrin tissue adhesives have found several practical uses in surgerysuch as cardiac surgery, vascular surgery, plastic surgery, andreconstructive surgery as hemostatic agents as well as adhesives. Theymay also be used for the closure of both skin grafts and local skinflaps. There are a variety of applicators available to deliver fibrintissue adhesives to the surgical field. The simplest is the sequentialdelivery of the first component and second component with two separatesyringes. A dual-syringe applicator can also be used. Fibrin tissueadhesives have the advantage that they are biocompatible andbiodegradable. They also show minimal tissue reactivity. However, fibrintissue adhesives have the disadvantage that they are very expensive. Inaddition, fibrin tissue adhesives are not easy to handle. For example,the two components of the fibrin tissue adhesive have to be transportedand stored deep frozen (e.g. at −20° C.). Thus, it is very importantthat the (distribution) cold chain is not interrupted. In addition,fibrin tissue adhesives have to be packed as two separate components toavoid fibrin clot formation before application. However, even aftermixing, the processing time is very short before the fibrin clotformation is completed.

Acrylate-based tissue adhesives such as cyanoacrylates were first usedin surgery 1959 when Coover (see Coover H W, Joyner F B, et al.“Chemistry and performance of cyanoacrylate adhesives.” J Soc Plast Eng1959; 15: 413-7) discovered their inherent adhesive properties.Cyanoacrylates include, but are not limited to, methyl-2-cyanoacrylate,buytl-2-cyanoacrylate, and octyl-2-cyanoacrylate. These compounds havetheir greatest utility in surgery such as facial plastic andreconstructive surgery as an alternative to traditional suture closure.They have also been used to close superficial wounds. In contrast tofibrin tissue adhesives, which rely on the interaction of endogenouscompounds, the cyanoacrylate tissue adhesives are synthetic compoundsthat do not naturally occur in the human or animal body. One method ofsynthesizing an alkyl cyanoacrylate monomer is by reacting alkylcyanoacetate with paraformaldehyde to form an intermediate compound.Heat applied to this intermediate compound causes depolymerization,resulting in an alkyl cyanoacrylate monomer liquid distillate (Toriumi DM, Raslan W F, Friedman M, et al. “Histotoxicity of cyanoacrylate tissueadhesives.” Arch Otolaryngol Head Neck Surg 1990; 116: 546-50).Acrylate-based tissue adhesives, e.g. cyanoacrylates, are less expensivethan fibroin tissue adhesives. However, acrylate-based tissue adhesives,e.g. cyanoacrylates, have been shown to be histotoxic when applied belowthe dermis. For example, when applied below the skin, cyanoacrylates mayinduce histotoxicity as a result of biodegradation of the polymer intocyanoacetate and formaldehyde. In addition, acrylate-based tissueadhesives have the disadvantage that they seal the treated area tight sothat cells can not enter this area to reconnect the separated tissues.

Thus, there is a need for tissue adhesives which overcome theafore-mentioned problems.

The inventors of the present invention surprisingly found thatself-assembling polypeptides, particularly silk polypeptides such asspider silk polypeptides, are ideal tissue adhesives as they meet thefollowing criteria: sufficient binding strength, uncomplicated andtime-independent application, tissue biocompatibility, no tissuereactivity such as allergic or inflammatory reactions, and reasonablecosts. It is also remarkable that the adhesive effect is achievedwithout enzymatic and/or chemical reactions as described forfibrin-based and acrylate-based tissue adhesives. Further, when used astissue adhesives, the self-assembling polypeptides, particularly silkpolypeptides such as spider silk polypeptides, do not form aninsurmountable barrier so that the glued sites can be entered and passedby cells to generate new tissue. Furthermore, the self-assemblingpolypeptides, particularly silk polypeptides such as spider silkpolypeptides, have the advantage that they are flexible which reduces oreven abolishes the tensile force acting on the affected area. Inaddition, in contrast to acrylate- and fibroin-based tissue adhesives,self-assembling polypeptides prevent or at least delay drying out of theglued tissues.

Systems for the recombinant production of self-assembling polypeptides,particularly silk polypeptides, e.g. spider silk polypeptides are knownin the art. Particularly, systems for the recombinant production ofspider silk polypeptides in E. coli have been developed in WO2006/008163 and WO 2006/002827. As an example, it is referred to WO2006/008163 (claiming priority of U.S. provisional application No.60/590,196). In this expression system, single building blocks(=modules) can be varied freely and can thus be adapted to therequirements of the specific case. Modules of this type are disclosedalso in Hummerich et al., “Primary structure elements of dragline silksand their contribution to protein solubility and assembly”, 2004,Biochemistry 43, 13604-13612. Further modules are described in WO2007/025719.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a self-assemblingpolypeptide for use as tissue adhesive.

In a second aspect, the present invention relates to the use of aself-assembling polypeptide as tissue adhesive.

In a third aspect, the present invention relates to the use of aself-assembling polypeptide to glue one or more cosmetic compounds onskin, mucosa, and/or hair.

In a fourth aspect, the present invention relates to a self-assemblingpolypeptide for use in gluing one or more pharmaceutical compounds ontissue, skin, mucosa, and/or hair.

In a fifth aspect, the present invention relates to an applicationcombination comprising

-   -   i) a self-assembling polypeptide, and    -   ii) a factor enhancing self-assembly        for use as tissue adhesive.

In a sixth aspect, the present invention relates to the use of anapplication combination comprising

-   -   i) a self-assembling polypeptide, and    -   ii) a factor enhancing self-assembly as tissue adhesive.

In a seventh aspect, the present invention relates to the use of anapplication combination comprising

-   -   i) a self-assembling polypeptide, and    -   ii) a factor enhancing self-assembly        to glue one or more cosmetic compounds on skin, mucosa, and/or        hair.

In an eight aspect, the present invention relates to an applicationcombination comprising

-   -   i) a self-assembling polypeptide, and    -   ii) a factor enhancing self-assembly        for use in gluing one or more pharmaceutical compounds on        tissue, skin, mucosa, and/or hair.

In a ninth aspect, the present invention relates to a self-assemblingpolypeptide for use as organ protection and/or isolation material.

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodology, protocols and reagents described herein as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims. Unless defined otherwise herein, all technicaland scientific terms used herein have the same meanings as commonlyunderstood by one of ordinary skill in the art.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, Leuenberger, H. G. W, Nagel, B. and Kölbl, H. eds.(1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, GenBank Accession Number sequence submissions etc.),whether supra or infra, is hereby incorporated by reference in itsentirety. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

In the following, the elements of the present invention will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and preferred embodiments should not be construed to limit thepresent invention to only the explicitly described embodiments. Thisdescription should be understood to support and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed and/or preferred elements. Furthermore, any permutationsand combinations of all described elements in this application should beconsidered disclosed by the description of the present applicationunless the context indicates otherwise.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integer or step.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural referents, unless the contentclearly dictates otherwise.

Residues in two or more polypeptides are said to “correspond” to eachother if the residues occupy an analogous position in the polypeptidestructures. It is well known in the art that analogous positions in twoor more polypeptides can be determined by aligning the polypeptidesequences based on amino acid sequence or structural similarities. Suchalignment tools are well known to the person skilled in the art and canbe, for example, obtained on the World Wide Web, e.g., ClustalW(www.ebi.ac.uk/clustalw) or Align(http://www.ebi.ac.uk/emboss/align/index.html) using standard settings,preferably for Align EMBOSS: needle, Matrix: Blosum62, Gap Open 10.0,Gap Extend 0.5.

Unless otherwise indicated, the terms “polypeptide” and “protein” areused interchangeably herein and mean any peptide-linked chain of aminoacids, regardless of length or post-translational modification.

As mentioned above, the inventors of the present invention surprisinglyfound that self-assembling polypeptides, particularly silk polypeptidessuch as spider silk polypeptides, are ideal tissue adhesives as theymeet the following criteria: sufficient binding strength, uncomplicatedand time-independent application, tissue biocompatibility, no tissuereactivity such as allergic or inflammatory reactions, and reasonablecosts. It is also remarkable that the adhesive effect is achievedwithout enzymatic and/or chemical reactions as described for fibrin andacrylate-based tissue adhesives. Further, when used as tissue adhesives,the self-assembling polypeptides, particularly silk polypeptides such asspider silk polypeptides, do not form an insurmountable barrier so thatthe glued sites can be entered and passed by cells to generate newtissue. Furthermore, the self-assembling polypeptides, particularly silkpolypeptides such as spider silk polypeptides, have the advantage thatthey are flexible which reduces or even abolishes the tensile forceacting on the effected area. In addition, in contrast to acrylate- andfibroin-based tissue adhesives, self-assembling polypeptides prevent orat least delay drying out of the glued tissues.

Thus, in a first aspect, the present invention relates to aself-assembling polypeptide for use as tissue adhesive.

The term “self-assembling polypeptide”, as used herein, refers to apolypeptide which can perform self-assembly. “Self-assembly” is a termused to describe a process in which a disordered system of pre-existingpolypeptides forms an organized structure or pattern as a consequence ofspecific, local interactions (e.g. van der Waals forces, hydrophobicinteractions, hydrogen bonds, and/or salt-bridges, etc.) among thepolypeptides themselves, without external direction or trigger althoughexternal factors might influence speed and nature of self-assembly. Thisparticularly means that when two or more disordered and/or unfoldedpolypeptides are brought into contact, they interact with each other andconsequently form a three dimensional structure. The change from adisordered system to an organised structure or pattern duringself-assembly is characterized by a transition from a fluid state to agelatinous and/or solid state and a corresponding increase in viscosity.The transition from a fluid state to a gelatinous state can bemonitored, for example, by measurement of light scattering, rheology, orCircular Dichroism (CD). These techniques are known to the skilledperson. The transition from a fluid state to a solid state can bemonitored, for example, using optical methods.

Preferably, the self-assembling polypeptide is biodegradable,biocompatible, non-immunogenic and/or non-inflammatory.

The “tissue adhesive (also designated as tissue sealant or tissueglue)”, as used herein, allows to connect, particularly reconnect,tissue layers, e.g. at least two tissue layers, with each other.Particularly, the tissue adhesive can provide a close, especiallyform-fit, connection between tissue layers, or in the event that thetissue layers are distant from each other, the tissue adhesive can fillthe gap between the tissue layers, replace the missing tissue layersand/or bridge the missing tissue layers. Preferably, the gap has a sizeof no more than 1 cm, more preferably of no more than 0.75 cm, even morepreferably of no more than 0.5 cm, most preferably of no more than 0.25cm, e.g. of no more than 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04,0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095,0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7,0.75, 0.8, 0.85, 0.9, 0.95, or 1 cm. Alternatively or additionally, the“tissue adhesive” allows to connect tissue layers to artificialsurfaces, e.g. of medical devices such as implants, or allows to connectartificial surfaces, e.g. of medical devices such as implants, to tissuelayers. Thereby, the medical devices such as implants can be embedded orincorporated in the tissue.

The tissue is preferably selected from the group consisting ofconnective tissue, muscle tissue, nervous tissue, epithelial tissue, andcombinations thereof, e.g. multiple (different) tissues. An organ, e.g.stomach, small intestine, large intestine, bowel, rectum, oesophagus,lung, spleen, brain, heart, kidney, liver, skin, glands such as lymphand thyroid glands, eye, or pancreas, is, for example, comprised ofmultiple (different) tissues.

The inventors of the present invention further surprisingly found thatthe adhesive effect can be enhanced when a self-assembling polypeptide,particularly a silk polypeptide such as spider silk polypeptide, is usedwhich further comprises a peptide having a cell adhesion mediatingprotein (CAMP) recognition sequence (e.g. RGD). This cell adhesionmediating protein (CAMP) recognition sequence (e.g. RGD) allows thebinding of the peptide to CAMP proteins (e.g. integrins) which arepresent on the surface of cells and which are involved in the binding toother cells and/or to the extracellular matrix (ECM) in a process calledcell adhesion. The surprising adhesive effect of RGD, as shown herein,is different to the effect known in the art that silk polypeptidescontaining RGD support cell growth. One remarkable difference betweenthis embodiment of the invention and the state of the art is that theadhesive effect is exclusively based on a strong molecule interactionbetween the recognition sequence and CAMP and, thus, occurs withinminutes or even faster whereas the cell adhesion effect previouslydescribed only occurs after days.

Thus, it is preferred that the self-assembling polypeptide furthercomprises at least one peptide, e.g. at least two peptides, particularlyat least two identical peptides, which is (are) capable of enhancing theadhesive effect. The term “peptide” means a short polymer formed fromthe linking, in a defined order, of α-amino acids. The link between oneamino acid residue and the next is called an amide bond or a peptidebond. Preferably, the at least one peptide, e.g. at least two peptides,particularly at least two identical peptides, which is (are) capable ofenhancing the adhesive effect

-   -   (i) is (are) composed of between 3 and 30 amino acids, more        preferably between 3 and 20 amino acids, and even more        preferably between 3 and 15 amino acids, e.g. 3, 4, 5, 6, 7, 8,        9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,        25, 26, 27, 28, 29, or 30 amino acids, and/or    -   (ii) is (are) (a) linear peptide(s), cyclic peptide(s), or        peptide analog(s).

It is particularly preferred that the at least one peptide, e.g. atleast two peptides, particularly at least two identical peptides, is(are) capable of enhancing the adhesive effect by at least 10%, morepreferably by at least 20%, even more preferably by at least 40%, andmost preferably by at least 60% or 80%, e.g. by at least 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80%. Tests to measurethe adhesive effect are described in the experimental section.

It is more preferred that the at least one peptide, e.g. at least twopeptides, particularly at least two identical peptides, which is (are)capable of enhancing the adhesive effect is (are) attached to theself-assembling polypeptide, e.g. to the N- and/or C-terminus of theself-assembling polypeptide.

Said attachment may occur via genetical fusion. For genetical fusion,for example, the peptide which is capable of enhancing the adhesiveeffect may be fused by genetic engineering to the self-assemblingpolypeptide.

Alternatively or additionally, said attachment may occur via chemicalcoupling, for example, via a covalent bond such as a peptide bond and/ornon-peptide bond, e.g. disulfide-bond. Said attachment may be directlyor indirectly, e.g. via a linker. The term “linker”, as used herein,refers to a moiety that connects the peptide which is capable ofenhancing the adhesive effect with the self-assembling polypeptidecovalently. It may be comprised at the N- and/or C-terminus of theself-assembling polypeptide. In this case, the linker may be designatedas TAG. Preferred herein are peptide linkers. This means that thepeptide linker is an amino acid sequence that connects the peptide whichis capable of enhancing the adhesive effect with the self-assemblingpolypeptide. The peptide linker may be connected to the peptide by apeptide or non-peptide bond and to the self-assembling polypeptide by apeptide or non-peptide bond. For example, the peptide linker may beconnected (i) to the peptide and to the self-assembling polypeptide viaa peptide bond, (ii) to the peptide and to the self-assemblingpolypeptide via a non-peptide bond, (iii) to the peptide via anon-peptide bond and to the self-assembling polypeptide via apeptide-bond, or (iv) to the peptide via a peptide-bond and to theself-assembling polypeptide via a non-peptide bond. When the peptidelinker is connected to the peptide via a non-peptide bond, this may bedone via a thiol-group of a cysteine (C) of the linker. In addition,when the peptide linker is connected to the peptide via a peptide-bond,this may be done via a ε-group of a lysine (K) residue of the linker.Typically, a peptide linker has a length of between 1 and 20 aminoacids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 amino acids. It is preferred that said linker comprises atleast one cysteine (C) or lysine (K) residue. It is also preferred thatthe amino sequence of the peptide linker is not immunogenic to humanbeings. For example, a linker having at least the amino acid sequenceGC, CG, GK or KG, or having at least the amino acid sequence GCG, GKG orany other peptide linker, e.g. GGCG (SEQ ID NO: 23), GCGG (SEQ ID NO:24), GGKG (SEQ ID NO: 27), or GKGG (SEQ ID NO: 28), can be used in thepresent invention. Also other linkers for the chemical coupling of twoprotein monomers, are known in the art and can be used.

Particularly, the at least one peptide, e.g. at least two peptides,particularly at least two identical peptides, can be attached to theself-assembling polypeptide or linker via a side group of an amino acid,e.g. via a thiol-group, amino-group, or carboxy-group, of said linker orself-assembling polypeptide. The attachment of the at least one peptide,e.g. at least two peptides, particularly at least two identicalpeptides, to the self-assembling polypeptide or linker can also takeplace via a reactive group (e.g. SMCC,Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) optionallycomprising a spacer moiety, such as an amino-carboxylic acid, inparticular an amino-carboxylic acid which comprises the amino group atthe ω-C-atom. The reactive group optionally comprising a spacer moietymay be coupled to the peptide, linker and/or self-assemblingpolypeptide. Preferably, the amino-carboxylic acid comprises between 2to 10 C-atoms, and more preferably between 4, 6, and 8 C-atoms, e.g. 2,3, 4, 5, 6, 7, 8, 9, or 10 C-atoms. More preferably, theamino-carboxylic acid is 6-amino-hexanoic acid. The term “reactivegroup”, as used herein, means any group which is capable of forming acovalent bond to a side group of an amino acid. Beyond that, the skilledperson knows how to connect the peptide with the self-assemblingpolypeptide.

In a preferred embodiment, the self-assembling polypeptide furthercomprises an amino terminal and/or a carboxy terminal TAG selected fromthe group consisting of

-   -   i) TAG^(CYS1) consisting of the amino acid sequence        GCGGGGGGSGGGG (SEQ ID NO: 35),    -   ii) TAG^(CYS2) consisting of the amino acid sequence GCGGGGGG        (SEQ ID NO: 36),    -   iii) TAG^(CYS3) consisting of the amino acid sequence        GCGGSGGGGSGGGG (SEQ ID NO: 37),    -   iv) TAG^(LYS1) consisting of the amino acid sequence        GKGGGGGGSGGGG (SEQ ID NO: 38), and    -   v) TAG^(LYS2) consisting of the amino acid sequence GKGGGGGG        (SEQ ID NO: 39).

Said TAG is preferably attached (e.g. covalently linked or coupled) tothe N- and/or C-terminus of the self-assembling polypeptide. Thethiol-group of the cysteine residue comprised in the above TAGs allowsthe coupling of the peptide to said TAGs and, thus, also to theself-assembling polypeptide. In a more preferred embodiment, the peptidewhich is capable of enhancing the adhesive effect is covalently coupledto the thiol-group of a cysteine residue of TAG^(CYS3).

Preferably, the adhesive effect is mediated via binding of the peptideto a cell adhesion mediating protein (CAMP). Said binding is preferablya non-covalent binding, particularly a non-covalent and reversiblebinding. The term “cell adhesion mediating protein (CAMP)”, as usedherein, refers to a protein located on the cell surface involved in thebinding with other cells and/or with the extracellular matrix (ECM) in aprocess called cell adhesion. Essentially, CAMPs help cells to stick toeach other and to their surroundings. These proteins are typicallytransmembrane receptors and are composed of three domains: anintracellular domain that interacts with the cytoskeleton, atransmembrane domain, and an extracellular domain that interacts eitherwith other CAMPs of the same kind (homophilic binding), or with otherCAMPs or the extracellular matrix (heterophilic binding). Particularlythe extracellular domain comprised in the CAMPs interacts with CAMPrecognition sequences.

The term “cell adhesion”, as used herein, refers to a mechanism whichallows the coupling of cells to adjacent cells (cell-cell adhesion)and/or the coupling of cells to the surrounding ECM (cell-ECM adhesion).Cell adhesion is the basis for the establishment and development ofmulticellular organisms. Cell adhesion processes are important for cellmorphogenesis and the development of tissues. Therefore, cell adhesionplays an important role during proliferation, migration, and/ordifferentiation. Particularly, the cell adhesion mechanism is a cellularprocess which includes wound healing, homeostasis, tumor growth, andmetastasis.

Most of the CAMPs belong to three protein families: the integrins, thecadherins, and the selectins. Thus, in preferred embodiments of thepresent invention, the CAMP is an integrin, a selectin, or a cadherin.

The term “integrins”, as used herein, refers to a family of receptorsthat mediate the attachment between a cell and the tissue that surroundsit, such as other cells and/or the extracellular matrix (ECM). Integrinsare calcium dependent. They are a family of heterophilic CAMPs.Integrins are heterodimers containing two distinct chains, called a(alpha) and β (beta) subunits. The different combinations of α and βsubunits result in a number of functional integrins. Integrin α/βheterodimers can be grouped into three subfamilies, namely β1-integrin,β2-integrin, and αv-integrin. Thus, for example, the integrin isselected from the group consisting of β1-integrin, β2-integrin, andαv-integrin.

The term “selectins (or cluster of differentiation 62/CD62 proteins)”,as used herein, refers to a family of single-chain transmembraneglycoproteins that share similar properties to C-type lectins due to arelated amino terminus and calcium-dependent binding.http://en.wikipedia.org/wiki/Selectin-cite note-robspath-1# citenote-robspath-1 Selectins bind to sugar moieties and so are consideredto be a type of lectin cell adhesion proteins that bind sugar polymers.They are a family of heterophilic CAMPs. Preferably, the selectin isselected from the group consisting of L-selectin, P-selectin, andE-selectin.

The term “cadherins (or calcium-dependent adhesion proteins)”, as usedherein, refers to a class of type-1 transmembrane proteins. They are afamily of homophilic CAMPs. Cadherins play important roles in celladhesion ensuring that cells within tissues are bound together. They aredependent on calcium (Ca²⁺) ions to function, hence their name.Preferably, cadherins are selected from the group consisting ofclassical cadherins, desmosomal cadherins, protocadherins, andunconventional/ungrouped cadherins. It is particularly preferred thatthe (classical) cadherin is selected from the group consisting ofE-cadherin, N-cadherin, and P-cadherin.

More preferably, the peptide which is capable of enhancing the adhesiveeffect comprises at least one CAMP recognition sequence, e.g. at leastone, two, three, or four CAMP recognition sequence(s). The at least oneCAMP recognition sequence particularly allows the binding of the peptidewhich is capable of enhancing the adhesive effect to the CAMP. Thus, inpreferred embodiments of the invention, the adhesive effect is mediatedvia binding of the peptide by its at least one CAMP recognition sequenceto the CAMP. Said binding is preferably a non-covalent binding,particularly a non-covalent and reversible binding.

CAMP recognition sequences occur naturally in CAMP-binding proteins,e.g. in fibronectin, fibrinogen, or vitronectin. Examples ofCAMP-binding proteins, their CAMP recognition sequences and CAMPs towhich they bind are listed in Table 1.

CAMP recognition sequences which may be used in the present inventioncomprise or consists of a RGD-containing module or a non-RGD-containingmodule.

The RGD sequence is the cell attachment site of a large number ofextracellular matrix (EM), blood, and cell surface proteins.Particularly, integrins recognize this sequence (see, for example,Ruoslahti, E. “RGD and other recognition sequences for integrins.”,Annual Review of Cell and Developmental Biology, 1996, Vol. 12, pages697-715.). The RGD sequence is, for example, naturally comprised in thebone sialoprotein, in collagen, decorsin, disintegrin, fibronectin,fibrinogen, fibrillin, prothrombin, thrombospondin, tenascin,vitronectin, or in the von Willebrand factor. Proteins with a CAMPrecognition sequence which differs from RGD are, for example, collagen,cytotactin/tenascin-C, epiligrin, factor X, α-Chain of fibrinogen,γ-Chain of fibrinogen, invasin, laminin, matrix metalloproteinase-2,neutrophil inhibitory factor, osteopontin, plasminogen, spectrin,tenascin, or VCAM-1.

The GER, GEN, and GEK sequences are, for example, cell attachment siteswhich are naturally comprised in collagens. Said sequences areparticularly used by collagen-binding integrins (see, for example,Raynal, N. et al., “Use of synthetic peptides to locate novel integrinα2β1-binding motifs in human collagen III.”, Journal of biologicalchemistry, 2006, Vol. 281, pages 3821-3831.) Further, the IDAPS sequenceis, for example, a cell attachment site which is naturally comprised infibronectin and the GPR and HHLGGAKQAGDV sequences are, for example,cell attachment sites which are naturally comprised in fibrinogen.Particularly, integrins recognize these sequences. Furthermore, theCDPGYIGSR sequence is, for example, a cell attachment site which isnaturally comprised in laminin, the AEIDGIEL sequence is, for example, acell attachment site which is naturally comprised in tenascin, the QIDSsequence is, for example, a cell attachment site which is naturallycomprised in VCAM-1, and the LDT sequence is, for example, a cellattachment site which is naturally comprised in MAdCAM-1.

TABLE 1 Examples of CAMP-binding proteins, their CAMP recognitionsequences and CAMPs to which they bind. CAMP-binding CAMP recognitionprotein CAMP sequence Bone sialoprotein Integrin RGD Collagen IntegrinRGD GFOGER (O = Hydroxyproline) Decorsin Integrin RGD DisintegrinIntegrin RGD Fibronectin Integrin RGD IDAPS Fibrinogen Integrin RGDα-Chain of fibrinogen Integrin GPR γ-Chain of fibrinogen IntegrinHHLGGAKQAGDV Laminin Integrin CDPGYIGSR MAdCAM-1 Integrin LDTProthrombin Integrin RGD Thrombospondin Integrin RGD Tenascin IntegrinRGD AEIDGIEL VCAM-1 Integrin QIDS Vitronectin Integrin RGD vonWillebrand factor Integrin RGD

In preferred embodiments, the CAMP recognition sequence comprises amodule containing or consisting of RGD, GER, GEK, GEN, IDAPS (SEQ ID NO:45) or variants thereof, GPR, HHLGGAKQAGDV (SEQ ID NO: 46) or variantsthereof, CDPGYIGSR (SEQ ID NO: 47) or variants thereof, AEIDGIEL (SEQ IDNO: 48) or variants thereof, QIDS (SEQ ID NO: 49), or LTD. In morepreferred embodiments, the CAMP recognition sequence comprises a modulecontaining or consisting of RGD.

As mentioned above, it is preferred that the peptide which is capable ofenhancing the adhesive effect comprises at least one CAMP recognitionsequence, e.g. at least one, two, three, or four CAMP recognitionsequence(s). Preferably, said CAMP recognition sequence comprises amodule containing or consisting of RGD, GER, GEK, GEN, IDAPS (SEQ ID NO:45) or variants thereof, GPR, HHLGGAKQAGDV (SEQ ID NO: 46) or variantsthereof, CDPGYIGSR (SEQ ID NO: 47) or variants thereof, AEIDGIEL (SEQ IDNO: 48) or variants thereof, QIDS (SEQ ID NO: 49), or LTD. Thus, forexample, the peptide may comprise (i) one, two, three, or fourRGD-containing modules, (ii) one, two, three, or four GER-containingmodules, (iii) one, two, three, or four GEK-containing modules, (iv)one, two, three, or four GEN-containing modules, (v) one modulecontaining RGD and one module containing GER, (vi) one module containingRGD and one module containing GEK, (vii) one module containing RGD andone module containing GEN, (viii) one module containing GER and onemodule containing GEK, (ix) one module containing GER and one modulecontaining GEN, (x) one module containing GEK and one module containingGEN, (xi) one module containing RGD, one module containing GER, and onemodule containing GEK, (xii) one module containing RGD, one modulecontaining GER, and one module containing GEN, (xiii) one modulecontaining RGD, one module containing GEK, and one module containingGEN, (xiv), one module containing GER, one module containing GEK, andone module containing GEN, and (xv) one module containing RGD, onemodule containing GER, one module containing GEK, and one modulecontaining GEN.

The above mentioned variants, i.e. IDAPS, HHLGGAKQAGDV, CDPGYIGSR, orAEIDGIEL variants, differ from the IDAPS, HHLGGAKQAGDV, CDPGYIGSR, orAEIDGIEL references (wild-types) from which they are derived by up to 1,2, 3, or 4 amino acid changes in the amino acid sequence (i.e.substitutions, additions, insertions, deletions, N-terminal truncationsand/or C-terminal truncations). Such variants can alternatively oradditionally be characterised by a certain degree of sequence identityto the references (wild-types) from which they are derived. Thus, theIDAPS, HHLGGAKQAGDV, CDPGYIGSR, or AEIDGIEL variants have a sequenceidentity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% to the respective IDAPS, HHLGGAKQAGDV,CDPGYIGSR, or AEIDGIEL references (wild-types). It is particularlypreferred that the sequence identity is at least 85% over the wholelength, is at least 85% over the whole length, is at least 90% over thewhole length, is at least 95% over the whole length, is at least 98%over the whole length, or is at least 99% over the whole length of therespective IDAPS, HHLGGAKQAGDV, CDPGYIGSR, or AEIDGIEL references(wild-types).

Fragments (or deletion variants) of IDAPS, HHLGGAKQAGDV, CDPGYIGSR, orAEIDGIEL have preferably a deletion of up to 1 or 2 amino acids at itsN-terminus and/or at its C-terminus. The deletion can also beinternally.

Additionally, the IDAPS, HHLGGAKQAGDV, CDPGYIGSR, or AEIDGIEL variantsor fragments are only regarded as a IDAPS, HHLGGAKQAGDV, CDPGYIGSR, orAEIDGIEL variants or fragments within the context of the presentinvention, if the modifications with respect to the amino acid sequenceon which the variants or fragments are based do not negatively affectthe ability of the peptide to bind to the CAMP. The skilled person canreadily assess whether the peptide comprising a modified CAMPrecognition sequence is still able to bind to the CAMP, e.g. byperforming protein binding studies. For example, the IsothermalTitration calorimetry (ITC) is a powerful analytical tool which measuresthe binding affinity and thermodynamics between any two biomolecules. Inaddition, the Surface Plasmon Resonance (SPR) based technology can beused for studying biomolecular interactions in real time.

In even more preferred embodiments, the CAMP recognition sequencecomprises a module containing a linear or cyclic RGD. A CAMP recognitionsequence which comprises a module containing a linear RGD is preferredin cases where the peptide is fused by genetic engineering to theself-assembling polypeptide. In contrast thereto, a CAMP recognitionsequence which comprises a module containing a cyclic RGD is preferredin cases where the peptide is fused by chemical coupling to theself-assembling polypeptide. In most preferred embodiments, the modulecontaining a linear RGD is selected from the group consisting of RGDS(SEQ ID NO: 50), GRGDS (SEQ ID NO: 51), GRGDY (SEQ ID NO: 52),GGSGGRGDSPG (SEQ ID NO: 53), RGDSPASSKP (SEQ ID NO: 54), andCGGNGEPRGDYRAY (SEQ ID NO: 55), and/or the module containing a cyclicRGD is selected from the group consisting of c(RGDfK), c(RGDfC), andc(RGDfE). Thereby, RGD stands for the amino acids arginine (Arg),glycine (Gly), and aspartic acid (Asp); f stands for D-phenylalanine(D-Phe); and K stands for lysine (Lys), C stands for cysteine (Cys) andE stands for glutamic acid (Glu), respectively.

Alternatively or additionally, the CAMP recognition sequence comprising

-   -   i) a module containing GER is selected from the group consisting        of GFOGER (SEQ ID NO: 56), GLOGER (SEQ ID NO: 57), GASGER (SEQ        ID NO: 58), GROGER (SEQ ID NO: 59), GMOGER (SEQ ID NO: 60),        GLSGER (SEQ ID NO: 61), and GAOGER (SEQ ID NO: 62),    -   ii) a module containing GEK is selected from the group        consisting of GFOGEK (SEQ ID NO: 63), GLOGEK (SEQ ID NO: 64),        GASGEK (SEQ ID NO: 65), GROGEK (SEQ ID NO: 66), GMOGEK (SEQ ID        NO: 67), GLSGEK (SEQ ID NO: 68), and GAOGEK (SEQ ID NO: 69), or    -   iii) a module containing GEN is selected from the group        consisting of GLOGEN (SEQ ID NO: 70) and GLKGEN (SEQ ID NO: 71).

The “O” in the above sequences refers to “hydroxyproline”.

In a preferred embodiment of the invention, the peptide which is capableof enhancing the adhesive effect comprises at least one CAMP recognitionsequence comprising or consisting of a module containing RGD, preferablya linear RGD, more preferably a linear RGD which is selected from thegroup consisting of RGDS (SEQ ID NO: 50), GRGDS (SEQ ID NO: 51), GRGDY(SEQ ID NO: 52), GGSGGRGDSPG (SEQ ID NO: 53), RGDSPASSKP (SEQ ID NO:54), and CGGNGEPRGDYRAY (SEQ ID NO: 55), or a cyclic RGD, morepreferably a cyclic RGD selected from the group consisting of c(RGDfK),c(RGDfC), and c(RGDfE), wherein the adhesive effect is mediated viabinding of said peptide to an integrin as cell adhesion mediatingprotein (CAMP). Said binding is preferably a non-covalent binding, morepreferably a non-covalent and reversible binding.

In another preferred embodiment of the invention, the peptide which iscapable of enhancing the adhesive effect comprises at least one CAMPrecognition sequence comprising or consisting of a module selected fromthe group consisting of (i) GER, preferably GFOGER (SEQ ID NO: 56),GLOGER (SEQ ID NO: 57), GASGER (SEQ ID NO: 58), GROGER (SEQ ID NO: 59),GMOGER (SEQ ID NO: 60), GLSGER (SEQ ID NO: 61), or GAOGER (SEQ ID NO:62), (ii) GEK, preferably GFOGEK (SEQ ID NO: 63), GLOGEK (SEQ ID NO:64), GASGEK (SEQ ID NO: 65), GROGEK (SEQ ID NO: 66), GMOGEK (SEQ ID NO:67), GLSGEK (SEQ ID NO: 68), or GAOGEK (SEQ ID NO: 69), and (iii) GEN,preferably GLOGEN (SEQ ID NO: 70) or GLKGEN (SEQ ID NO: 71), wherein theadhesive effect is mediated via binding of said peptide to an integrinas cell adhesion mediating protein (CAMP). Said binding is preferably anon-covalent binding, more preferably a non-covalent and reversiblebinding. The “O” in the above sequences refers to “hydroxyproline”.

Preferably, the self-assembling polypeptide is selected from the groupconsisting of a silk polypeptide, an elastin, a collagen, and a keratin.The silk polypeptide may be expressed in a recombinant, e.g. microbial,insect, plant, or mammalian expression system, i.e. separated from itsnatural milieu, (recombinant silk polypeptide), or may be harvested fromnatural sources, e.g. spider, silk worm, bee, mussel, or fly larvae. Thesilk polypeptide may be isolated or purified. In particular, a “purifiedsilk polypeptide” or an “isolated silk polypeptide” is free orsubstantially free of cellular material, production/fermentationremnants, and/or other contaminating proteins from the cell or tissuesource from which the protein is purified or isolated. The language“substantially free of cellular material” includes preparations of asilk polypeptide in which the silk polypeptide is separated fromcellular components of the cells from which it is recombinantlyproduced. A silk polypeptide that is “substantially free” of cellularmaterial, production/fermentation remnants, and/or other contaminatingproteins from the cell or tissue source from which the protein ispurified or isolated includes preparations of silk polypeptides havingless than about 30%, 20%, 10%, 5%, 1%, or 0.1% (by dry weight) ofcontaminating protein and/or less than about 30%, 20%, 10%, 5%, 1%, or0.1% (by dry weight) of contaminating lipid, DNA or salt.

It is more preferred that the silk polypeptide is a recombinant silkpolypeptide. It is also more preferred that the silk polypeptide is aspider silk polypeptide, even more preferably a major ampullate silkpolypeptide such as a dragline silk polypeptide, a minor ampullate silkpolypeptide, or a flagelliform silk polypeptide of an orb-web spider(e.g. Araneidae or Araneoids), an insect silk polypeptide, a musselbyssus silk polypeptide, or a mixture thereof. The orb-web spider may beselected from the group consisting of Araneus diadematus, Nephilaclavipes, and Latrodectus hesperus. The insect silk polypeptide may beof Lepidoptera, particularly Bombycidae such as Bombyx mori. The insectsilk polypeptide may also be of Hymenoptera, particularly Apoidea suchas Anthophila.

It is even more preferred that the silk polypeptide is a recombinantspider silk polypeptide, most preferably a recombinant major ampullatesilk polypeptide such as a recombinant dragline silk polypeptide, arecombinant minor ampullate silk polypeptide, or a recombinantflagelliform silk polypeptide of an orb-web spider (e.g. Araneidae orAraneoids), a recombinant insect silk polypeptide, a recombinant musselbyssus silk polypeptide, or a mixture thereof. The orb-web spider may beselected from the group consisting of Araneus diadematus, Nephilaclavipes, and Latrodectus hesperus. The recombinant insect silkpolypeptide may be of Lepidoptera, particularly Bombycidae such asBombyx mori. The insect silk polypeptide may also be of Hymenoptera,particularly Apoidea such as Anthophila.

It is also (alternatively or additionally) preferred that the silkpolypeptide comprises, essentially consists of, or consists of at leasttwo identical repetitive units. Said at least two identical repetitiveunits comprise or consists of identical copies of amino acid sequencesof naturally occurring silk polypeptides or of variations of amino acidsequences of naturally-occurring silk polypeptides or of combinations ofboth.

The term a “repetitive unit”, as used herein, refers to a region whichcorresponds in amino acid sequence to a region that comprises orconsists of at least one peptide motif (e.g. AAAAAA (SEQ ID NO: 13) orGPGQQ (SEQ ID NO: 4)) that repetitively occurs within a naturallyoccurring silk polypeptide (e.g. MaSpI, ADF-3, ADF-4, or Flag) (i.e.identical amino acid sequence) or to an amino acid sequencesubstantially similar thereto (i.e. variational amino acid sequence). Inthis regard “substantially similar” means a degree of amino acididentity of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even99.9%, preferably over the whole length of the respective referencenaturally occurring amino acid sequence.

A “repetitive unit” having an amino acid sequence which is“substantially similar” to a corresponding amino acid sequence within anaturally occurring silk polypeptide (i.e. wild-type repetitive unit) isalso similar with respect to its functional properties, e.g. a silkpolypeptide comprising the “substantially similar repetitive unit” stillhas the ability to act as adhesive, particularly to function as tissueadhesive. The skilled person can readily assess whether the silkpolypeptide comprising a “substantially similar repetitive unit” isstill acting as adhesive, particularly is still functioning as tissueadhesive, for example, by conducting the adhesive or pulling test asdescribed in the experimental section.

A “repetitive unit” having an amino acid sequence which is “identical”to the amino acid sequence of a naturally occurring silk polypeptide canbe, for example, a portion of a silk polypeptide corresponding to one ormore peptide motifs of MaSp I (SEQ ID NO: 43) MaSp II (SEQ ID NO: 44),ADF-3 (SEQ ID NO: 1) and/or ADF-4 (SEQ ID NO: 2). A “repetitive unit”having an amino acid sequence which is “substantially similar” to theamino acid sequence of a naturally occurring silk polypeptide can be,for example, a portion of a silk polypeptide corresponding to one ormore peptide motifs of MaSpI (SEQ ID NO: 43) MaSpII (SEQ ID NO: 44),ADF-3 (SEQ ID NO: 1) and/or ADF-4 (SEQ ID NO: 2), but having one or moreamino acid substitution(s) at (a) specific amino acid position(s).

The term, a “repetitive unit”, as used herein, does not include thenon-repetitive hydrophilic amino acid domain generally thought to bepresent at the amino terminus and/or carboxyl terminus of naturallyoccurring silk polypeptides.

The term a “repetitive unit”, as used herein, further refers to an aminoacid sequence with a length of 3 to 200 amino acids, or 5 to 150 aminoacids, preferably with a length of 10 to 100 amino acids, or 15 to 80amino acids and more preferably with a length of 18 to 60, or 20 to 40amino acids. For example, the repetitive unit according to the presentinvention can have a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115,120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185,190, 195, or 200 amino acids. Most preferably, the repetitive unitaccording to the invention consists of 3, 4, 5, 6, 7, 8, 9, 10, 12, 15,18, 20, 24, 27, 28, 30, 34, 35, or 39 amino acids.

It should be noted that the terms “repetitive unit” and “repeat unit”can interchangeable be used in the context of the present invention.

In particularly preferred embodiments, the silk polypeptide is apolypeptide with an amino acid sequence which comprises or consists ofat least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, preferably at least 95%and most preferably 100% of multiple copies of one identical repetitiveunit (e.g. A₂, Q₆, or C₁₆, wherein the items 2, 6, or 16 represent thenumber of repetitive units) or multiple copies of two or more differentrepetitive units (e.g. (AQ)₂₄, or (AQ)₁₂C₁₆). Said silk polypeptide canfurther be modified by adding an artificial tag to facilitate thedetection or purification of said protein (e.g. T7 tag).

The repetitive unit of the silk polypeptide can comprise or consist ofan amino acid sequence of any region that comprises or consists of atleast one peptide motif that repetitively occurs within a naturallyoccurring silk polypeptide known to one skilled in the art. Preferably,the repetitive unit of the silk polypeptide comprises or consists of anamino acid sequence of a region that comprises or consists of at leastone peptide motif that repetitively occurs within an arthropod silkpolypeptide, more preferably within a spider silk polypeptide, or aninsect silk polypeptide. The repetitive unit of the silk polypeptide canalso comprise or consist of an amino acid sequence of a region thatcomprises or consists of at least one peptide motif that repetitivelyoccurs within a mussel silk polypeptide.

It is preferred that the spider silk repetitive unit comprises orconsists of an amino acid sequence of a region that comprises orconsists of at least one peptide motif that repetitively occurs within anaturally occurring major ampullate silk polypeptide (MaSp), such as adragline silk polypeptide, a minor ampullate silk polypeptide (MiSp), ora flagelliform (FLAG) silk polypeptide. Most preferably, the repetitiveunit comprises or consists of an amino acid sequence of a region thatcomprises or consists of at least one peptide motif that repetitivelyoccurs within a naturally occurring dragline silk polypeptide orflagelliform silk polypeptide.

It is also preferred that the insect silk repetitive unit comprises orconsists of an amino acid sequence of a region that comprises orconsists of at least one peptide motif that repetitively occurs within anaturally occurring silk polypeptide of Lepidoptera. More preferably,the insect silk repetitive unit comprises or consists of an amino acidsequence of a region that comprises or consists of at least one peptidemotif that repetitively occurs within a naturally occurring insect silkpolypeptide of Bombycidae, most preferably of Bombyx mori.

The term “consensus sequence”, as used herein, refers to an amino acidsequence which contains amino acids which frequently occur in a certainposition (e.g. “G”) and wherein, other amino acids which are not furtherdetermined are replaced by the place holder “X”.

Preferably, the silk polypeptide comprises, essentially consists of, orconsists of at least two identical repetitive units each comprising atleast one, preferably one, consensus sequence selected from the groupconsisting of:

-   -   i) GPGXX (SEQ ID NO: 3), wherein X is any amino acid, preferably        in each case independently selected from A, S, G, Y, P, and Q;    -   ii) GGX, wherein X is any amino acid, preferably in each case        independently selected from Y, P, R, S, A, T, N and Q, more        preferably in each case independently selected from Y, P and Q;    -   iii) A_(x), wherein x is an integer from 5 to 10;    -   iv) GGRPSDTYG (SEQ ID NO: 18); and    -   v) GGRPSSSYG (SEQ ID NO: 19).

More preferably, the above mentioned silk polypeptide has a molecularweight of at least 5 kDa, e.g. at least 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 kDa. Thus, in a more preferred embodiment,the self-assembling polypeptide has a molecular weight of at least 5kDa, e.g. at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 kDa, and comprises at least two identical repetitive unitseach comprising at least one consensus sequence selected from the groupconsisting of:

-   -   i) GPGXX (SEQ ID NO: 3), wherein X is any amino acid, preferably        in each case independently selected from A, S, G, Y, P, and Q;    -   ii) GGX, wherein X is any amino acid, preferably in each case        independently selected from Y, P, R, S, A, T, N and Q, more        preferably in each case independently selected from Y, P and Q;    -   iii) A_(x), wherein x is an integer from 5 to 10;    -   iv) GGRPSDTYG (SEQ ID NO: 18); and    -   v) GGRPSSSYG (SEQ ID NO: 19).

The iterated (peptide) motifs GPGXX (SEQ ID NO: 3) and GGX, i.e. glycinerich motifs, provide flexibility to the silk polypeptide and thus, tothe thread formed from the silk protein containing said motifs. Indetail, the iterated GPGXX (SEQ ID NO: 3) motif forms (3-turn spiralstructures, which imparts elasticity to the silk polypeptide. Majorampullate and flagelliform silks both have a GPGXX (SEQ ID NO: 3) motif.The iterated GGX motif is associated with a helical structure havingthree amino acids per turn and is found in most spider silks. The GGXmotif may provide additional elastic properties to the silk. Theiterated polyalanine A_(x) (peptide) motif forms a crystalline (3-sheetstructure that provides strength to the silk polypeptide. (WO03/057727). The GGRPSDTYG (SEQ ID NO: 18) and GGRPSSSYG (SEQ ID NO: 19)(peptide) motifs have been selected from Resilin (WO 08/155304). Resilinis an elastomeric protein found in most arthropods (arthropoda). It islocated in specialised regions of the cuticle, providing low stiffnessand high strength (Elvin et al., Nature (473): 999-1002, 2005).

Thus, in a preferred embodiment of the present invention, the silkpolypeptide comprises or consists of repetitive units each comprising atleast one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, or 9), preferably one, aminoacid sequence selected from the group consisting of GPGAS (SEQ ID NO:5), GPGSG (SEQ ID NO: 6), GPGGY (SEQ ID NO: 7), GPGGP (SEQ ID NO: 8),GPGGA (SEQ ID NO: 9), GPGQQ (SEQ ID NO: 4), GPGGG (SEQ ID NO: 10), GPGQG(SEQ ID NO: 40), and GPGGS (SEQ ID NO: 11). In a further preferredembodiment of the present invention, the silk polypeptide comprises orconsists of repetitive units each comprising at least one (e.g. 1, 2, 3,4, 5, 8, 7, or 8), preferably one, amino acid sequence selected from thegroup consisting of GGY, GGP, GGA, GGR, GGS, GGT, GGN, and GGQ. In anadditionally preferred embodiment of the present invention, the silkpolypeptide comprises or consists of repetitive units each comprising atleast one (e.g. 1, 2, 3, 4, 5, or 6), preferably one, amino acidsequence selected from the group consisting of AAAAA (SEQ ID NO: 12),AAAAAA (SEQ ID NO: 13), AAAAAAA (SEQ ID NO: 14), AAAAAAAA (SEQ ID NO:15), AAAAAAAAA (SEQ ID NO: 16), and AAAAAAAAAA (SEQ ID NO: 17).

In another preferred embodiment of the invention, the silk polypeptidecomprises or consists of repetitive units each comprising at least one(e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or 25), preferably one, amino acid sequence selectedfrom the group consisting of GPGAS (SEQ ID NO: 5), GPGSG (SEQ ID NO: 6),GPGGY (SEQ ID NO: 7), GPGGP (SEQ ID NO: 8), GPGGA (SEQ ID NO: 9), GPGQQ(SEQ ID NO: 4), GPGGG (SEQ ID NO: 10), GPGQG (SEQ ID NO: 40), GPGGS (SEQID NO: 11), GGY, GGP, GGA, GGR, GGS, GGT, GGN, GGQ, AAAAA (SEQ ID NO:12), AAAAAA (SEQ ID NO: 13), AAAAAAA (SEQ ID NO: 14), AAAAAAAA (SEQ IDNO: 15), AAAAAAAAA (SEQ ID NO: 16), AAAAAAAAAA (SEQ ID NO: 17),GGRPSDTYG (SEQ ID NO: 18) and GGRPSSSYG (SEQ ID NO: 19).

Most preferably, the silk polypeptide comprises, essentially consistsof, or consists of repetitive units, which comprise or consist of

-   -   i) GPGAS (SEQ ID NO: 5), AAAAAA (SEQ ID NO: 13), GGY, and GPGSG        (SEQ ID NO: 6) as amino acid sequence, preferably in this order,    -   ii) AAAAAAAA (SEQ ID NO: 15), GPGGY (SEQ ID NO: 7), GPGGY (SEQ        ID NO: 7), and GPGGP (SEQ ID NO: 8) as amino acid sequence,        preferably in this order,    -   iii) GPGQQ (SEQ ID NO: 4), GPGQQ (SEQ ID NO: 4), GPGQQ (SEQ ID        NO: 4) and GPGQQ (SEQ ID NO: 4) as amino acid sequence,    -   iv) GPGGA (SEQ ID NO: 9), GGP, GPGGA (SEQ ID NO: 9), GGP, GPGGA        (SEQ ID NO: 9), and GGP as amino acid sequence, preferably in        this order,    -   v) AAAAAAAA (SEQ ID NO: 15), GPGQG (SEQ ID NO: 40), and GGR as        amino acid sequence, preferably in this order,    -   vi) AAAAAAAA (SEQ ID NO: 15), GPGGG (SEQ ID NO: 10), GGR, GGN,        and GGR as amino acid sequence, preferably in this order,    -   vii) GGA, GGA, GGA, GGS, GGA, and GGS as amino acid sequence,        preferably in this order, and/or    -   viii) GPGGA (SEQ ID NO: 9), GPGGY (SEQ ID NO: 7), GPGGS (SEQ ID        NO: 11), GPGGY (SEQ ID NO: 7), GPGGS (SEQ ID NO: 11), and GPGGY        (SEQ ID NO: 7) as amino acid sequence, preferably in this order.

It should be noted that at least two of the repetitive units comprisedin the silk polypeptides mentioned above are identical repetitive units.

Preferably, the silk polypeptide comprises, essentially consists of, orconsists of between 2 to 80 repetitive units, between 3 to 80 repetitiveunits, or between 4 to 60 repetitive units, more preferably between 8 to48 repetitive units, or between 10 to 40 repetitive units and mostpreferably between 16 to 32 repetitive units, e.g. 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80repetitive units, each comprising at least one, preferably one,consensus sequence selected from the group consisting of:

-   -   i) GPGXX (SEQ ID NO: 3), wherein X is any amino acid, preferably        in each case independently selected from A, S, G, Y, P, and Q;    -   ii) GGX, wherein X is any amino acid, preferably in each case        independently selected from Y, P, R, S, A, T, N and Q, more        preferably in each case independently selected from Y, P and Q;    -   iii) A_(x), wherein x is an integer from 5 to 10.    -   iv) GGRPSDTYG (SEQ ID NO: 18); and    -   v) GGRPSSSYG (SEQ ID NO: 19).

More preferably, the above mentioned silk polypeptide has a molecularweight of at least 5 kDa, e.g. at least 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 kDa. As mentioned above, at least two ofthe repetitive units comprised in the silk polypeptide are identicalrepetitive units.

Thus, the silk polypeptide preferably comprises or consists of between 2to 80 repetitive units, between 3 to 80 repetitive units, or between 4to 60 repetitive units, more preferably between 8 to 48 repetitiveunits, or between 10 to 40 repetitive units and most preferably between16 to 32 repetitive units, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 repetitive units,each comprising at least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25), preferablyone, amino acid sequence selected from the group consisting of GPGAS(SEQ ID NO: 5), GPGSG (SEQ ID NO: 6), GPGGY (SEQ ID NO: 7), GPGGP (SEQID NO: 8), GPGGA (SEQ ID NO: 9), GPGQQ (SEQ ID NO: 4), GPGQG (SEQ ID NO:40), GPGGG (SEQ ID NO: 10), GPGGS (SEQ ID NO: 11), GGY, GGP, GGA, GGR,GGS, GGT, GGN, GGQ, AAAAA (SEQ ID NO: 12), AAAAAA (SEQ ID NO: 13),AAAAAAA (SEQ ID NO: 14), AAAAAAAA (SEQ ID NO: 15), AAAAAAAAA (SEQ ID NO:16), AAAAAAAAAA (SEQ ID NO: 17), GGRPSDTYG (SEQ ID NO: 18) and GGRPSSSYG(SEQ ID NO: 19).

Most preferably, the silk polypeptide comprises, essentially consistsof, or consists of

-   -   i) repetitive units which comprise or consist of GPGAS (SEQ ID        NO: 5), AAAAAA (SEQ ID NO: 13), GGY, and GPGSG (SEQ ID NO: 6) as        amino acid sequence, preferably in this order,    -   ii) repetitive units which comprise or consist of AAAAAAAA (SEQ        ID NO: 15), GPGGY (SEQ ID NO: 7), GPGGY (SEQ ID NO: 7), and        GPGGP (SEQ ID NO: 8) as amino acid sequence, preferably in this        order,    -   iii) repetitive units which comprise or consist of GPGQQ (SEQ ID        NO: 4), GPGQQ (SEQ ID NO: 4), GPGQQ (SEQ ID NO: 4) and GPGQQ        (SEQ ID NO: 4) as amino acid sequence,    -   iv) repetitive units which comprise or consist of GPGGA (SEQ ID        NO: 9), GGP, GPGGA (SEQ ID NO: 9), GGP, GPGGA (SEQ ID NO: 9),        and GGP as amino acid sequence, preferably in this order,    -   v) repetitive units which comprise or consist of AAAAAAAA (SEQ        ID NO: 15), GPGQG (SEQ ID NO: 40), and GGR as amino acid        sequence, preferably in this order,    -   vi) repetitive units which comprise or consist of AAAAAAAA (SEQ        ID NO: 15), GPGGG (SEQ ID NO: 10), GGR, GGN, and GGR as amino        acid sequence, preferably in this order,    -   vii) repetitive units which comprise or consist of GGA, GGA,        GGA, GGS, GGA, and GGS as amino acid sequence, preferably in        this order, and/or    -   viii) repetitive units which comprise or consist of GPGGA (SEQ        ID NO: 9), GPGGY (SEQ ID NO: 7), GPGGS (SEQ ID NO: 11), GPGGY        (SEQ ID NO: 7), GPGGS (SEQ ID NO: 11), and GPGGY (SEQ ID NO: 7)        as amino acid sequence, preferably in this order.

It should be noted that at least two of the repetitive units comprisedin the silk polypeptides mentioned above are identical repetitive units.

Preferably, the silk polypeptide comprises, essentially consists of, orconsists of

-   -   i) (GPGXX)_(n) (SEQ ID NO: 3) as a repetitive unit, wherein X is        any amino acid, preferably in each case independently selected        from A, S, G, Y, P, and Q and n is 2, 3, 4, 5, 6, 7, 8, or 9;    -   ii) (GGX)_(n) as a repetitive unit, wherein X is any amino acid,        preferably in each case independently selected from Y, P, R, S,        A, T, N and Q, more preferably in each case independently        selected from Y, P and Q, and n is 2, 3, 4, 5, 6, 7, or 8;        and/or    -   iii) (A_(x))_(n) as a repetitive unit, wherein x is an integer        from 5 to 10 and n is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

More preferably, the above mentioned silk polypeptide has a molecularweight of at least 5 kDa, e.g. at least 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 kDa. As mentioned above, at least two ofthe repetitive units comprised in the silk polypeptides are identicalrepetitive units.

It is further preferred that the repetitive units are independentlyselected from module A (SEQ ID NO: 20), module C (SEQ ID NO: 21), moduleQ (SEQ ID NO: 22), module S (SEQ ID NO: 25), module R (SEQ ID NO: 26),or variants thereof (i.e. module A variants, module C variants, module Qvariants, module S variants, or module R variants). Modules A (SEQ IDNO: 20) and Q (SEQ ID NO: 22) are based on the amino acid sequence ofADF-3 of the spider Araneus diadematus. Module C (SEQ ID NO: 21) isbased on the amino acid sequence of ADF-4 of the spider Araneusdiadematus. Modules S (SEQ ID NO: 25) and R (SEQ ID NO: 26) are based onResilin (Arthropoda) (WO 2008/155304).

Thus, in a preferred embodiment, the repetitive units of the silkpolypeptide consist of module A: GPYGPGASAAAAAAGGYGPGSGQQ (SEQ ID NO:20), module C: GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 21),module Q: GPGQQGPGQQGPGQQGPGQQ (SEQ ID NO: 22), module S:PGSSAAAAAAAASGPGQGQGQGQGQGGRPSDTYG (SEQ ID NO: 25), module R:SAAAAAAAAGPGGGNGGRPSDTYGAPGGGNGGRPSSSYG (SEQ ID NO: 26), or variantsthereof.

It is further particularly preferred that the silk polypeptidecomprises, essentially consists of, or consists of between 2 to 80repetitive units, between 3 to 80 repetitive units, or between 4 to 60repetitive units, more preferably between 8 to 48 repetitive units, orbetween 10 to 40 repetitive units and most preferably between 16 to 32repetitive units, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 repetitive units, which areindependently selected from module A (SEQ ID NO: 20), module C (SEQ IDNO: 21), module Q (SEQ ID NO: 22), module S (SEQ ID NO: 25), module R(SEQ ID NO: 26), or variants thereof (i.e. module A variants, module Cvariants, module Q variants, module S variants, or module R variants).It should be noted that at least two of the repetitive units comprisedin the silk polypeptide are identical repetitive units (modules).

Thus, in a (particularly) preferred embodiment, the silk polypeptidecomprises, essentially consists of, or consists of (i) repetitiveunit(s) consisting of module A and/or repetitive unit(s) consisting ofmodule A variants, (ii) repetitive unit(s) consisting of module C and/orrepetitive unit(s) consisting of module C variants, (iii) repetitiveunit(s) consisting of module Q and/or repetitive unit(s) consisting ofmodule Q variants, (iv) (a) repetitive unit(s) consisting of module Aand repetitive unit(s) consisting of module Q, (b) repetitive unit(s)consisting of module A and repetitive unit(s) consisting of module Qvariants, (c) repetitive unit(s) consisting of module A variants andrepetitive unit(s) consisting of module Q, (d) repetitive unit(s)consisting of module A variants and repetitive unit(s) consisting ofmodule Q variants, (v) (a) repetitive unit(s) consisting of module A andrepetitive unit(s) consisting of module C, (b) repetitive unit(s)consisting of module A and repetitive unit(s) consisting of module Cvariants, (c) repetitive unit(s) consisting of module A variants andrepetitive unit(s) consisting of module C, (d) repetitive unit(s)consisting of module A variants and repetitive unit(s) consisting ofmodule C variants, (vi) (a) repetitive unit(s) consisting of module Cand repetitive unit(s) consisting of module Q, (b) repetitive unit(s)consisting of module C and repetitive unit(s) consisting of module Qvariants, (c) repetitive unit(s) consisting of module C variants andrepetitive unit(s) consisting of module Q, (d) repetitive unit(s)consisting of module C variants and repetitive unit(s) consisting ofmodule Q variants, or (vii) (a) repetitive unit(s) consisting of moduleA, repetitive unit(s) consisting of module Q and repetitive unit(s)consisting of module C, (b) repetitive unit(s) consisting of module A,repetitive unit(s) consisting of module Q and repetitive unit(s)consisting of module C variants, (c) repetitive unit(s) consisting ofmodule A, repetitive unit(s) consisting of module Q variants andrepetitive unit(s) consisting of module C, (d) repetitive unit(s)consisting of module A variants, repetitive unit(s) consisting of moduleQ and repetitive unit(s) consisting of module C, (e) repetitive unit(s)consisting of module A, repetitive unit(s) consisting of module Qvariants and repetitive unit(s) consisting of module C variants, (f)repetitive unit(s) consisting of module A variants, repetitive unit(s)consisting of module Q variants and repetitive unit(s) consisting ofmodule C, (g) repetitive unit(s) consisting of module A variants,repetitive unit(s) consisting of module Q and repetitive unit(s)consisting of module C variants, (h) repetitive unit(s) consisting ofmodule A variants, repetitive unit(s) consisting of module Q variantsand repetitive unit(s) consisting of module C variants.

The modules A, C, Q, S, R, or variants thereof (i.e. module A variants,module C variants, module Q variants, module S variants, or module Rvariants) can also be combined or concatenated with each other in anycombination and in any number of each, i.e. module (repetitive unit) Acan be combined with module (repetitive unit) Q (i.e. combination AQ),module (repetitive unit) C can be combined with module (repetitive unit)Q (i.e. combination CQ), module (repetitive unit) Q can be combined withmodule (repetitive unit) A and with module (repetitive unit) Q (i.e.combination QAQ), module (repetitive unit) A can be combined with module(repetitive unit) A and with module (repetitive unit) Q (i.e.combination AAQ), etc., under the proviso that the silk polypeptidecomprises or consists of at least two repetitive units which areidentical. For example, the silk polypeptide can comprise or consist ofA_(n), (AA)_(n), (AQ)_(n), (QA)_(n), Q_(n), (QQ)_(n), (QAQ)_(n),(AQA)_(n), C_(n), (CC)_(n), (CCC)_(n), (CQ)_(n), (QC)_(n), (QCQ)_(n),(CQC)_(n), (AA)_(n)Q_(n), (QQ)_(n)A_(n), (AAA)_(n)Q_(n), (QQQ)_(n)A_(n),(AQQ)_(n), (QQA)_(n), S_(n), R_(n), (SS)_(n), (SR)_(n), (RS)_(n), or(RR)_(n), wherein n is at least 2, preferably 4, 8, 9, 10, 12, 16, 20,24, or 32. In case that the silk polypeptide consists of (AQ)₁₂, it isnoted that module (repetitive unit) A is 12 times present and module(repetitive unit) Q is also 12 times present in the silk polypeptide andthat, thus, the silk polypeptide consists of 24 modules (repetitiveunits). The arrangement of the modules (repeat units) of a silkpolypeptide consisting of (AQ)₁₂ is as follows:AQAQAQAQAQAQAQAQAQAQAQAQ. Further, in case that the silk polypeptide ofthe modules (repeat units) of a silk polypeptide consists of (QAQ)₈, itis noted that module (repeat unit) A is 8 times present and module(repetitive unit) Q is 16 times present in the silk polypeptide andthat, thus, the silk polypeptide consists of 24 modules (repetitiveunits). The arrangement of the modules (repeat units) of a silkpolypeptide consisting of (QAQ)₈ is as follows:QAQQAQQAQQAQQAQQAQQAQQAQ. The silk polypeptide can also comprise orconsist of (A*Q)_(n), (AQ*)_(n), (A*Q*)_(n), (Q*A)_(n), (QA*)_(n),(Q*A*)_(n), (QAQ*)_(n), (QA*Q)_(n), (Q*AQ)_(n), (QA*Q*)_(n),(Q*A*Q)_(n), (Q*AQ*)_(n), (Q*A*Q*)_(n), (AQA*)_(n), (AQ*A)_(n),(A*QA)_(n), (AQ*A*)_(n), (A*Q*A)_(n), (A*QA*)_(n), (A*Q*A*)_(n), whereinn is at least 2, preferably 4, 8, 9, 10, 12, 16, 20, 24, or 32 andwherein * indicates a module variant, i.e. module A or Q variant.

The terms “combined with each other” or “concatenated with each other”,as used herein, mean that the modules (repetitive units) are directlycombined or concatenated with each other, or mean that the modules(repetitive units) are combined or concatenated with each other via oneor more spacer amino acids. Thus, in one embodiment, the modules(repetitive units) comprised in the silk polypeptide are directlycombined or concatenated with each other. In another embodiment, themodules (repetitive units) comprised in the silk polypeptide arecombined or concatenated with each other via one or more spacer aminoacids, preferably via 1 to 25 or 1 to 20 spacer amino acids, morepreferably via 1 to 15 or 1 to 10 spacer amino acids, and mostpreferably, via 1 to 5 spacer amino acids, e.g. via 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25spacer amino acids. Said spacer amino acid may be any amino acidnaturally occurring in proteins. Preferably, said spacer amino acid isnot proline. It is preferred that the spacer amino acid contains acharged group(s). Preferably, the spacer amino acid containing a chargedgroup(s) is independently selected from the group consisting ofaspartate, glutamate, histidine, and lysine. Said spacer amino acidshould be an amino acid which does not negatively affect the ability ofa silk polypeptide to act as adhesive, particularly to function astissue adhesive. Further, said spacer amino acid should be an amino acidwhich does not cause steric hindrance, e.g. an amino acid having a smallsize such as lysine and cysteine. In more preferred embodiments, thesilk polypeptide comprises modules which are directly combined with eachother and modules which are combined with each other via 1 to 25 or 1 to20 spacer amino acids, more preferably via 1 to 15 or 1 to 10 spaceramino acids, and most preferably, via 1 to 5 spacer amino acids, e.g.via 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, or 25 spacer amino acids.

A module A, C, Q, S, or R variant differs from the reference (wild-type)module A, C, Q, S, or R from which it is derived by up to 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid changes in the aminoacid sequence (i.e. substitutions, additions, insertions, deletions,N-terminal truncations and/or C-terminal truncations). Such a modulevariant can alternatively or additionally be characterised by a certaindegree of sequence identity to the reference (wild-type) module fromwhich it is derived. Thus, a module A, C, Q, S, or R variant has asequence identity of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% oreven 99.9% to the respective reference (wild-type) module A, C, Q, S, orR. Preferably, the sequence identity is over a continuous stretch of atleast 10, 15, 18, 20, 24, 27, 28, 30, 34, 35, or more amino acids,preferably over the whole length of the respective reference (wild-type)module A, C, Q, S, or R.

It is particularly preferred that the sequence identity is at least 80%over the whole length, is at least 85% over the whole length, is atleast 90% over the whole length, is at least 95% over the whole length,is at least 98% over the whole length, or is at least 99% over the wholelength of the respective reference (wild-type) module A, C, Q, S, or R.It is further particularly preferred that the sequence identity is atleast 80% over a continuous stretch of at least 10, 15, 18, 20, 24, 28,or 30 amino acids, is at least 85% over a continuous stretch of at least10, 15, 18, 20, 24, 28, or 30 amino acids, is at least 90% over acontinuous stretch of at least 10, 15, 18, 20, 24, 28, or 30 aminoacids, is at least 95% over a continuous stretch of at least 10, 15, 18,20, 24, 28, or 30 amino acids, is at least 98% over a continuous stretchof at least 10, 15, 18, 20, 24, 28, or 30 amino acids, or is at least99% over a continuous stretch of at least 10, 15, 18, 20, 24, 28, or 30amino acids of the respective reference (wild-type) module A, C, Q, S,or R.

A fragment (or deletion variant) of module A, C, Q, S, or R haspreferably a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, or 15 amino acids at its N-terminus and/or at its C-terminus.The deletion can also be internally.

Additionally, the module A, C, Q, S, or R variant or fragment is onlyregarded as a module A, C, Q, S, or R variant or fragment within thecontext of the present invention, if the modifications with respect tothe amino acid sequence on which the variant or fragment is based do notnegatively affect the ability of the silk polypeptide to act asadhesive, particularly to function as tissue adhesive. The skilledperson can readily assess whether the silk polypeptide comprising amodule A, C, Q, S, or R variant or fragment is still acting as adhesive,particularly to function as tissue adhesive, for example, by conductingthe adhesive or pulling test as described in the experimental section.

It is more preferred that the repetitive units are independentlyselected from module A^(C) (SEQ ID NO: 29), module A^(K) (SEQ ID NO:30), module C^(C) (SEQ ID NO: 31), module C^(K1) (SEQ ID NO: 32), moduleC (SEQ ID NO: 33) or module C^(KC) (SEQ ID NO: 34). The modules A^(C)(SEQ ID NO: 29), A^(K) (SEQ ID NO: 30), C^(C) (SEQ ID NO: 31), C^(K1)(SEQ ID NO: 32), C^(K2) (SEQ ID NO: 33) and C^(KC) (SEQ ID NO: 34) arevariants of the module A which is based on the amino acid sequence ofADF-3 of the spider Araneus diadematus and of module C which is based onthe amino acid sequence of ADF-4 of the spider Araneus diadematus (WO2007/025719). In module A^(C) (SEQ ID NO: 29) the amino acid S (serine)at position 21 has been replaced by the amino acid C (cysteine), inmodule A^(K) (SEQ ID NO: 30) the amino acid S at position 21 has beenreplaced by the amino acid K (lysine), in module C^(C) (SEQ ID NO: 31)the amino acid S at position 25 has been replaced by the amino acid 25by C, in module C^(K1) (SEQ ID NO: 32) the amino acid S at position 25has been replaced by the amino acid K, in module C^(K2) (SEQ ID NO: 33)the amino acid E (glutamate) at position 20 has been replaced by theamino acid K, and in module C^(KC) (SEQ ID NO: 34) the amino acid E atposition 20 has been replaced by the amino acid K and the amino acid Sat position 25 has been replaced by the amino acid C (WO 2007/025719).Thus, in a more preferred embodiment, the repetitive units in the silkpolypeptide consist of module A^(C): GPYGPGASAAAAAAGGYGPGCGQQ (SEQ IDNO: 29), module A^(K): GPYGPGASAAAAAAGGYGPGKGQQ (SEQ ID NO: 30), moduleC^(C): GSSAAAAAAAASGPGGYGPENQGPCGPGGYGPGGP (SEQ ID NO: 31), moduleC^(K1): GSSAAAAAAAASGPGGYGPENQGPKGPGGYGPGGP (SEQ ID NO: 32), moduleC^(K2): GSSAAAAAAAASGPGGYGPKNQGPSGPGGYGPGGP (SEQ ID NO: 33), or moduleC^(KC): GSSAAAAAAAASGPGGYGPKNQGPCGPGGYGPGGP (SEQ ID NO: 34).

It is even more preferred that the silk polypeptide comprises,essentially consists of, or consists of between 2 to 80 repetitiveunits, between 3 to 80 repetitive units, or between 4 to 60 repetitiveunits, preferably between 8 to 48 repetitive units, or between 10 to 40repetitive units and most preferably between 16 to 32 repetitive units,e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79 or 80 repetitive units, which are independentlyselected from module A^(C) (SEQ ID NO: 29), module A^(K) (SEQ ID NO:30), module C^(C) (SEQ ID NO: 31), module C^(K1) (SEQ ID NO: 32), moduleC^(K2) (SEQ ID NO: 33) or module C^(KC) (SEQ ID NO: 34). It should benoted that at least two of the repetitive units comprised in the silkpolypeptide are identical repetitive units (modules).

It is most preferred that the silk polypeptide comprises, essentiallyconsists of, or consists of between 2 to 80 repetitive units, between 3to 80 repetitive units or between 4 to 60 repetitive units, preferablybetween 8 to 48 repetitive units, or between 10 to 40 repetitive unitsand most preferably between 16 to 32 repetitive units, e.g. 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79 or 80 repetitive units, which are independently selected from moduleA (SEQ ID NO: 20) or variants thereof, module C (SEQ ID NO: 21) orvariants thereof, module Q (SEQ ID NO: 22) or variants thereof, module S(SEQ ID NO: 25) or variants thereof, module R (SEQ ID NO: 26) orvariants thereof, module A^(C) (SEQ ID NO: 29), module A^(K) (SEQ ID NO:30), module C^(C) (SEQ ID NO: 31), module C^(K1) (SEQ ID NO: 32), moduleC^(K2) (SEQ ID NO: 33), or module C^(KC) (SEQ ID NO: 34). Again, itshould be noted that at least two of the repetitive units comprised inthe silk polypeptide are identical repetitive units (modules).

The modules A^(K), C^(C), C^(K1), C^(K2) and C^(KC) can also be combinedwith the modules A, C, Q, S, or R, i.e. module (repetitive unit) A^(K)can be combined with module (repetitive unit) C (i.e. combinationA^(K)C), or module (repetitive unit) C^(C) can be combined with module(repetitive unit) C (i.e. combination C^(C)C), etc., under the provisothat the silk polypeptide comprises or consists of at least tworepetitive units which are identical. Thus, the silk polypeptide canalso comprise or consist of the modules (AQA^(K))_(n), (QA^(K))_(n),(QA^(K)Q)_(n), (A^(K)QA)_(n), (A^(K)QA^(K))_(n), (CC^(C))_(n),(CC^(C)C)_(n), (C^(C)C^(C)C)_(n), (CC^(C)C^(C))_(n), (C^(C)Q)_(n),(QC^(C))_(n), (QC^(C)Q)_(n), (C^(C)QC)_(n), (CQC^(C))_(n),(C^(C)QC^(C))_(n), (CC^(K1))_(n), (C^(K1)C)_(n), (C^(K1)CC)_(n),(CC^(K1)C)_(n), (C^(KC)C^(KC)C)_(n), (CC^(KC)C^(KC))_(n), (C^(KC)Q)_(n),(QC^(KC))_(n), (QC^(KC)Q)_(n), (A^(K)C ^(K1)Q)_(n) (QC^(K2) A^(K))_(n),or (C^(K1) C^(K2)C)_(n), wherein n is at least 2, preferably 4, 5, 6, 7,8, 10, 12, 16, or 20. As to the terms “combined with each other” or“concatenated with each other”, it is referred to the definitionsprovided above. For example, the silk polypeptide comprises or consistsof the modules C₁₆C^(C), C^(C)C₁₆, C₈C^(C)C₈, C₈C^(C) ₈, C^(C) ₈C₈,C₄C^(C) ₈C₄, C^(C) ₄C₈C^(C) ₄, C^(C)(AQ)₂₄, or (AQ)₂₄C^(C).

The silk polypeptide can further comprise at least one non-repetitive(NR) unit, e.g. at least 1, 2, 3, 4, 5, 6, or more NR unit(s),preferably one NR unit. In the context of the present invention, theterm “non-repetitive (NR) unit” refers to a region of amino acidspresent in a naturally occurring silk polypeptide that displays noobvious repetition pattern (non-repetitive unit or NR unit). Preferably,the amino acid sequence of the non-repetitive unit corresponds to anon-repetitive amino acid sequence of naturally occurring draglinepolypeptides, preferably of ADF-3 (SEQ ID NO: 1) or ADF-4 (SEQ ID NO:2), or to an amino acid sequence substantially similar thereto. Theamino acid sequence of the non-repetitive unit may also correspond to anon-repetitive amino acid sequence of black widow. More preferably, theamino acid sequence of the non-repetitive unit corresponds to anon-repetitive carboxy terminal amino acid sequence of naturallyoccurring dragline polypeptides, preferably of ADF-3 (SEQ ID NO: 1) orADF-4 (SEQ ID NO: 2), or to an amino acid sequence substantially similarthereto. Even more preferably, the amino acid sequence of thenon-repetitive unit corresponds to a non-repetitive carboxy terminalamino acid sequence of ADF-3 (SEQ ID NO: 1) which comprises amino acids513 through 636, or of ADF-4 (SEQ ID NO: 2) which comprises amino acids302 through 410, or to an amino acid sequence substantially similarthereto.

In this regard “substantially similar” means a degree of amino acididentity of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even99.9%, preferably over 20, 30, 40, 50, 60, 70, 80 or more amino acids,more preferably over the whole length of the respective referencenon-repetitive (carboxy terminal) amino acid sequence of naturallyoccurring dragline polypeptides, preferably of ADF-3 (SEQ ID NO: 1) orADF-4 (SEQ ID NO: 2). A “non-repetitive unit” having an amino acidsequence which is “substantially similar” to a correspondingnon-repetitive (carboxy terminal) amino acid sequence within a naturallyoccurring dragline polypeptide (i.e. wild-type non-repetitive (carboxyterminal) unit), preferably within ADF-3 (SEQ ID NO: 1) or ADF-4 (SEQ IDNO: 2), is also similar with respect to its functional properties, e.g.a silk polypeptide comprising the “substantially similar non-repetitiveunit” still has the ability to act as adhesive, particularly to functionas tissue adhesive. The skilled person can readily assess whether thesilk polypeptide comprising the “substantially similar non-repetitiveunit” is still acting as adhesive, particularly is still functioning astissue adhesive, for example, by conducting the adhesive or pulling testas described in the experimental section.

Most preferably, the non-repetitive (NR) unit is NR3 (SEQ ID NO: 41) orvariants thereof, NR4 (SEQ ID NO: 42) or variants thereof, NR5 (SEQ IDNO: 76) or variants thereof, or NR6 (SEQ ID NO: 77) or variants thereof.The NR3 (SEQ ID NO: 41) unit is based on the amino acid sequence ofADF-3 of the spider Araneus diadematus and the NR4 (SEQ ID NO: 42) unitis based on the amino acid sequence of ADF-4 of the spider Araneusdiadematus (WO 2006/008163). In addition, the NR5 (SEQ ID NO: 76) unitand the NR6 (SEQ ID NO: 77) unit is derived from Latrodectus hesperus.

A NR3, NR4, NR5, or NR6 unit variant differs from the reference NR3 (SEQID NO: 41), NR4 (SEQ ID NO: 42), NR5 (SEQ ID NO: 76), or NR6 (SEQ ID NO:77) unit from which it is derived by up to 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 amino acid changesin the amino acid sequence (i.e. exchanges, insertions, deletions,N-terminal truncations and/or C-terminal truncations). Such a NR3, NR4,NR5, or NR6 unit variant can alternatively or additionally becharacterised by a certain degree of sequence identity to the referenceNR3, NR4, NR5, or NR6 unit from which it is derived. Thus, a NR3, NR4,NR5, or NR6 unit variant has a sequence identity of at least 50%, 55%,60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% oreven 99.9% to the respective reference NR3, NR4, NR5, or NR6 unit.Preferably, the sequence identity is over a continuous stretch of atleast 10, 20, 30, 40, 50, 60, 70, 80, 90, or more amino acids,preferably over the whole length of the respective reference NR3, NR4,NR5, or NR6 unit.

It is particularly preferred that the sequence identity is at least 80%over the whole length, is at least 85% over the whole length, is atleast 90% over the whole length, is at least 95% over the whole length,is at least 98% over the whole length, or is at least 99% over the wholelength of the respective reference NR3, NR4, NR5, or NR6 unit. It isfurther particularly preferred that the sequence identity is at least80% over a continuous stretch of at least 20, 30, 40, 50, 60, 70, or 80amino acids, is at least 85% over a continuous stretch of at least 20,30, 40, 50, 60, 70, or 80 amino acids, is at least 90% over a continuousstretch of at least 20, 30, 40, 50, 60, 70, or 80 amino acids, is atleast 95% over a continuous stretch of at least 20, 30, 40, 50, 60, 70,or 80 amino acids, is at least 98% over a continuous stretch of at least20, 30, 40, 50, 60, 70, or 80 amino acids, or is at least 99% over acontinuous stretch of at least 20, 30, 40, 50, 60, 70, or 80 amino acidsof the respective reference NR3, NR4, NR5, or NR6 unit.

A fragment (or deletion variant) of a NR3, NR4, NR5, or NR6 unit haspreferably a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50,55, or 60 amino acids at its N-terminus and/or at its C-terminus. Thedeletion can also be internally.

Additionally, the NR3, NR4, NR5, or NR6 unit variant or fragment is onlyregarded as a NR3, NR4, NR5, or NR6 unit variant or fragment within thecontext of the present invention, if the modifications with respect tothe amino acid sequence on which the variant or fragment is based do notnegatively affect the ability of a silk polypeptide to act as adhesive,particularly to function as tissue adhesive. The skilled person canreadily assess whether the silk polypeptide comprising a NR3, NR4, NR5,or NR6 unit variant or fragment is still acting as adhesive,particularly to function as tissue adhesive, for example, by conductingthe adhesive or pulling test as described in the experimental section.

Preferably, the silk polypeptide is selected from the group consistingof ADF-3 (SEQ ID NO: 1) or variants thereof, ADF-4 (SEQ ID NO: 2) orvariants thereof, MaSp I (SEQ ID NO: 43) or variants thereof, MaSp II(SEQ ID NO: 44) or variants thereof, (C) m, (C)_(m)NR_(z),NR_(z)(C)_(m), (AQ)_(n), (AQ)_(n)NR_(z), NR_(z)(AQ)_(n), (QAQ)_(o),NR_(z)(QAQ)_(o), (QAQ)_(o)NR_(z), wherein m is an integer of 8 to 48(i.e. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, or 48), n is an integer of 6 to 24 (i.e. 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24), o is aninteger of 8 to 16 (i.e. 8, 9, 10, 11, 12, 13, 14, 15, or 16), z is aninteger of 1 to 3 (i.e. 1, 2, or 3), and NR stands for a non-repetitiveunit. The above mentioned formulas are defined by one of the following:In the formula

-   -   i) (C)_(m), a “m” number of C modules, namely 8 to 48 C modules,        represented by the amino acid sequence according to SEQ ID NO:        21, are combined with each other,    -   ii) (C)_(m)NR_(z), a “m” number of C modules, namely 8 to 48 C        modules, represented by the amino acid sequence according to SEQ        ID NO: 21, are combined with each other, wherein said C modules        are further combined with a “z” number of non-repetitive (NR)        units, namely 1 to 3 non-repetitive (NR) units, e.g. the        non-repetitive (NR) units NR3 represented by the amino acid        sequence according to SEQ ID NO: 41, NR4 represented by the        amino acid sequence according to SEQ ID NO: 42, NR5 represented        by the amino acid sequence according to SEQ ID NO: 76, or NR6        represented by the amino acid sequence according to SEQ ID NO:        77,    -   iii) NR_(z)(C)_(m), a “z” number of non-repetitive (NR) units,        namely 1 to 3 non-repetitive (NR) units, e.g. the non-repetitive        (NR) units NR3 represented by the amino acid sequence according        to SEQ ID NO: 41, NR4 represented by the amino acid sequence        according to SEQ ID NO: 42, NR5 represented by the amino acid        sequence according to SEQ ID NO: 76, or NR6 represented by the        amino acid sequence according to SEQ ID NO: 77, is present (z=1)        or are combined with each other (z=2 or 3), wherein said        non-repetitive (NR) unit(s) is (are) further combined with a “m”        number of C modules, namely 8 to 48 C modules, represented by        the amino acid sequence according to SEQ ID NO: 21,    -   iv) (AQ)_(n), a “n” number of A and Q module combinations,        namely 6 to 24 A and Q module combinations, wherein module A is        represented by the amino acid sequence according to SEQ ID NO:        20 and module Q is represented by the amino acid sequence        according to SEQ ID NO: 22, are combined with each other,    -   v) (AQ)_(n)NR_(z), a “n” number of A and Q module combinations,        namely 6 to 24 A and Q module combinations, wherein module A is        represented by the amino acid sequence according to SEQ ID NO:        20 and module Q is represented by the amino acid sequence        according to SEQ ID NO: 22, are combined with each other, and        wherein said A and Q module combinations are further combined        with a “z” number of non-repetitive (NR) units, namely 1 to 3        non-repetitive (NR) units, e.g. the non-repetitive (NR) units        NR3 represented by the amino acid sequence according to SEQ ID        NO: 41, NR4 represented by the amino acid sequence according to        SEQ ID NO: 42, NR5 represented by the amino acid sequence        according to SEQ ID NO: 76, or NR6 represented by the amino acid        sequence according to SEQ ID NO: 77,    -   vi) NR_(z)(AQ)_(n), a “z” number of non-repetitive (NR) units,        namely 1 to 3 non-repetitive (NR) units, e.g. the non-repetitive        (NR) units NR3 represented by the amino acid sequence according        to SEQ ID NO: 41, NR4 represented by the amino acid sequence        according to SEQ ID NO: 42, NR5 represented by the amino acid        sequence according to SEQ ID NO: 76, or NR6 represented by the        amino acid sequence according to SEQ ID NO: 77, is present (z=1)        or are combined with each other (z=2 or 3), wherein said        non-repetitive (NR) unit(s) is (are) further combined with a “n”        number of A and Q module combinations, namely 6 to 24 A and Q        module combinations, wherein module A is represented by the        amino acid sequence according to SEQ ID NO: 20 and module Q is        represented by the amino acid sequence according to SEQ ID NO:        22,    -   vii) (QAQ)_(o), a “o” number of Q, A and Q module combinations,        namely 8 to 16 Q, A and Q module combinations, wherein module Q        is represented by an amino acid sequence according to SEQ ID NO:        22 and module A is represented by the amino acid sequence        according to SEQ ID NO: 20, are combined with each other,    -   viii) (QAQ)_(o)NR_(z), a “o” number of Q, A and Q module        combinations, namely 8 to 16 Q, A and Q module combinations,        wherein module Q is represented by an amino acid sequence        according to SEQ ID NO: 22 and module A is represented by the        amino acid sequence according to SEQ ID NO: 20, are combined        with each other, and wherein said Q, A and Q module combinations        are further combined with a “z” number of non-repetitive (NR)        units, namely 1 to 3 non-repetitive (NR) units, e.g. the        non-repetitive (NR) units NR3 represented by the amino acid        sequence according to SEQ ID NO: 41, NR4 represented by the        amino acid sequence according to SEQ ID NO: 42, NR5 represented        by the amino acid sequence according to SEQ ID NO: 76, or NR6        represented by the amino acid sequence according to SEQ ID NO:        77, and    -   ix) NR_(z)(QAQ)_(o), a “z” number of non-repetitive (NR) units,        namely 1 to 3 non-repetitive (NR) units, e.g. the non-repetitive        (NR) units NR3 represented by the amino acid sequence according        to SEQ ID NO: 41, NR4 represented by the amino acid sequence        according to SEQ ID NO: 42, NR5 represented by the amino acid        sequence according to SEQ ID NO: 76, or NR6 represented by the        amino acid sequence according to SEQ ID NO: 77, is present (z=1)        or are combined with each other (z=2 or 3), wherein said        non-repetitive (NR) unit(s) is (are) further combined with a “o”        number of Q, A and Q module combinations, namely 8 to 16 Q, A        and Q module combinations, wherein module Q is represented by an        amino acid sequence according to SEQ ID NO: 22 and module A is        represented by the amino acid sequence according to SEQ ID NO:        20.

More preferably, the silk polypeptide is C₁₆NR4, C₃₂NR4, (AQ)₁₂NR3,(AQ)₂₄NR3, (AQ)₁₂, (AQ)₂₄, C₁₆, C₃₂, NR4C₁₆NR4, NR4C₃₂NR4, NR3C₁₆NR3,NR3C₃₂NR3, NR4 (AQ)₁₂NR4, NR4(AQ)₂₄NR4, NR3(AQ)₁₂NR3, NR3(AQ)₂₄NR3,(QAQ)₈ or (QAQ)₁₆.

An ADF-3, ADF-4, MaSp I or MaSp II variant differs from the reference(wild-type) ADF-3 (SEQ ID NO: 1), ADF-4 (SEQ ID NO: 2), MaSp I (SEQ IDNO: 43) or MaSp II (SEQ ID NO: 44) polypeptide from which it is derivedby up to 150 (up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 110, 120, 130, 140, or 150) amino acid changes in the aminoacid sequence (i.e. substitutions, insertions, deletions, N-terminaltruncations and/or C-terminal truncations). Such a variant canalternatively or additionally be characterised by a certain degree ofsequence identity to the reference (wild-type) polypeptide from which itis derived. Thus, an ADF-3, ADF-4, MaSp I or MaSp II variant has asequence identity of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% oreven 99.9% to the respective reference (wild-type) ADF-3, ADF-4, MaSp Ior MaSp II polypeptide. Preferably, the sequence identity is over acontinuous stretch of at least 20, 25, 30, 35, 40, 50, 60, 70, 80, 90,100, 120, 150, 180, 200, 250, 300, 350, 400, or more amino acids,preferably over the whole length of the respective reference (wild-type)ADF-3, ADF-4, MaSp I or MaSp II polypeptide.

It is particularly preferred that the sequence identity is at least 80%over the whole length, is at least 85% over the whole length, is atleast 90% over the whole length, is at least 95% over the whole length,is at least 98% over the whole length, or is at least 99% over the wholelength of the respective reference (wild-type) ADF-3, ADF-4, MaSp I orMaSp II polypeptide. It is further particularly preferred that thesequence identity is at least 80% over a continuous stretch of at least20, 30, 50, 100, 150, 200, 250, or 300 amino acids, is at least 85% overa continuous stretch of at least 20, 30, 50, 100, 150, 200, 250, or 300amino acids, is at least 90% over a continuous stretch of at least 20,30, 50, 100, 150, 200, 250, or 300 amino acids, is at least 95% over acontinuous stretch of at least 20, 30, 50, 100, 150, 200, 250, or 300amino acids, is at least 98% over a continuous stretch of at least 20,30, 50, 100, 150, 200, 250, or 300 amino acids, or is at least 99% overa continuous stretch of at least 20, 30, 50, 100, 150, 200, 250, or 300amino acids of the respective reference (wild-type) ADF-3, ADF-4, MaSp Ior MaSp II polypeptide.

A fragment (or deletion variant) of the ADF-3 (SEQ ID NO: 1) polypeptidehas preferably a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,120, 150, 170, 200, 220, 250, 270, 300, 320, 350, 370, 400, 420, 450,470, 500, 520, 550, 570, 600, or 610 amino acids at its N-terminusand/or at its C-terminus. The deletion can also be internally.

A fragment (or deletion variant) of the ADF-4 (SEQ ID NO: 2) polypeptidehas preferably a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,120, 150, 170, 200, 220, 250, 270, 300, 320, 330, 340, 350, 360, 370,380, or 390 amino acids at its N-terminus and/or at its C-terminus. Thedeletion can also be internally.

A fragment (or deletion variant) of the MaSp I (SEQ ID NO: 43)polypeptide has preferably a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 620, 640,660, 670, 680, or 690 amino acids at its N-terminus and/or at itsC-terminus. The deletion can also be internally.

A fragment (or deletion variant) of the MaSp II (SEQ ID NO: 44)polypeptide has preferably a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 520, 540, 560, or 570amino acids at its N-terminus and/or at its C-terminus. The deletion canalso be internally.

Additionally, the ADF-3, ADF-4, MaSp I or MaSp II variant or fragment isonly regarded as an ADF-3, ADF-4, MaSp I or MaSp II variant or fragmentwithin the context of the present invention, if the modifications withrespect to the amino acid sequence on which the variant or fragment isbased do not negatively affect the ability of the silk polypeptide toact as adhesive, particularly to function as tissue adhesive. Theskilled person can readily assess whether the silk polypeptidecomprising a NR or NR4 unit variant or fragment is still acting asadhesive, particularly to function as tissue adhesive, for example, byconducting the adhesive or pulling test as described in the experimentalsection.

In a preferred embodiment of the invention, the silk polypeptide,preferably the spider silk polypeptide, more preferably major ampullatesilk polypeptide such as dragline silk polypeptide, minor ampullate silkpolypeptide, or flagelliform silk polypeptide of an orb-web spider (e.g.Araneidae or Araneoids), the insect silk polypeptide, or the musselbyssus silk polypeptide comprises, preferably attached (e.g. covalentlylinked or coupled) to the N- and/or C-terminus, a peptide capable ofenhancing the adhesive effect which comprises at least one CAMPrecognition sequence comprising or consisting of a module containingRGD, preferably a linear RGD, more preferably a linear RGD which isselected from the group consisting of RGDS (SEQ ID NO: 50), GRGDS (SEQID NO: 51), GRGDY (SEQ ID NO: 52), GGSGGRGDSPG (SEQ ID NO: 53),RGDSPASSKP (SEQ ID NO: 54), and CGGNGEPRGDYRAY (SEQ ID NO: 55), or acyclic RGD, more preferably a cyclic RGD selected from the groupconsisting of c(RGDfK), c(RGDfC), and c(RGDfE).

In a more preferred embodiment of the invention, the silk polypeptide,preferably the spider silk polypeptide, more preferably major ampullatesilk polypeptide such as dragline silk polypeptide, minor ampullate silkpolypeptide, or flagelliform silk polypeptide of an orb-web spider (e.g.Araneidae or Araneoids), the insect silk polypeptide, or the musselbyssus silk polypeptide comprises, preferably attached (e.g. covalentlylinked or coupled) to the N- and/or C-terminus, more preferably attached(e.g. covalently linked or coupled) to the C-terminus, a peptide capableof enhancing the adhesive effect which comprises at least one CAMPrecognition sequence comprising or consisting of a module containingGGSGGRGDSPG (SEQ ID NO: 53) (see, for example, FIG. 1B).

In another more preferred embodiment of the invention, the silkpolypeptide, preferably the spider silk polypeptide, more preferablymajor ampullate silk polypeptide such as dragline silk polypeptide,minor ampullate silk polypeptide, or flagelliform silk polypeptide of anorb-web spider (e.g. Araneidae or Araneoids), the insect silkpolypeptide, or the mussel byssus silk polypeptide comprises an aminoterminal and/or a carboxy terminal TAG, preferably an amino terminalTAG, selected from the group consisting of TAG^(CYS1) (SEQ ID NO: 35),TAG^(CYS2) (SEQ ID NO: 36), TAG^(CYS3) (SEQ ID NO: 37), TAG^(LYS1) (SEQID NO: 38) and TAG^(LYS2) (SEQ ID NO: 39), preferably TAG^(CYS3) (SEQ IDNO: 37), and further comprises a peptide capable of enhancing theadhesive effect which comprises at least one CAMP recognition sequencecomprising or consisting of a module containing c(RGDfK), c(RGDfC), orc(RGDfE), preferably c(RGDfK). Said TAG(s) is (are) preferably attached(e.g. covalently linked or coupled) to the N-terminus and/or C-terminusof said silk polypeptide, and/or said peptide is preferably attached(e.g. covalently linked or coupled) to said TAG(s). It is particularlypreferred that said peptide is covalently coupled to the thiol group ofa cysteine residue comprised in the amino terminal and/or carboxyterminal TAG selected from the group consisting of TAG^(CYS1) (SEQ IDNO: 35), TAG^(CYS2) (SEQ ID NO: 36), and TAG^(CYS3) (SEQ ID NO: 37)(see, for example, FIG. 1A).

In another preferred embodiment of the invention, the silk polypeptide,preferably the spider silk polypeptide, more preferably major ampullatesilk polypeptide such as a dragline silk polypeptide, minor ampullatesilk polypeptide, or flagelliform silk polypeptide of an orb-web spider(e.g. Araneidae or Araneoids), the insect silk polypeptide, or themussel byssus silk polypeptide comprises, preferably attached to the N-and/or C-terminus, a peptide capable of enhancing the adhesive effectwhich comprises at least one CAMP recognition sequence comprising orconsisting of a module selected from the group consisting of (i) GER,preferably GFOGER (SEQ ID NO: 56), GLOGER (SEQ ID NO: 57), GASGER (SEQID NO: 58), GROGER (SEQ ID NO: 59), GMOGER (SEQ ID NO: 60), GLSGER (SEQID NO: 61), or GAOGER (SEQ ID NO: 62), (ii) GEK, preferably GFOGEK (SEQID NO: 63), GLOGEK (SEQ ID NO: 64), GASGEK (SEQ ID NO: 65), GROGEK (SEQID NO: 66), GMOGEK (SEQ ID NO: 67), GLSGEK (SEQ ID NO: 68), or GAOGEK(SEQ ID NO: 69), and (iii) GEN, preferably GLOGEN (SEQ ID NO: 70) orGLKGEN (SEQ ID NO: 71). The “0” in the above sequences refers to“hydroxyproline”.

The above mentioned adhesive effect is preferably mediated via binding(e.g. non-covalent binding, particularly non-covalent and reversiblebinding) of said peptide to an integrin as cell adhesion mediatingprotein (CAMP). Additionally or alternatively, the above mentioned silkpolypeptides are recombinant silk polypeptides.

The self-assembling polypeptide, as described herein, may be provided inseveral preferential compositions including, but not limited to, solidcompositions such as powdery compositions, liquid compositions such asaqueous compositions, e.g. aqueous solutions, emulsions, or suspensions,aerosols such as dry or liquid aerosols, pump sprays, and pastycompositions. These compositions comprising the self-assemblingpolypeptide may additionally comprise one or more pharmaceuticalcompounds such as therapeutic or diagnostic compounds.

The self-assembling polypeptide, as described herein, may further beprovided as film, gel, particularly hydrogel, foam, mesh, scaffold,patch, nonwoven, or layer, particularly covering or coating layer. Thefilm, gel, particularly hydrogel, foam, mesh, scaffold, path, nonwoven,or layer, particularly covering or coating layer, comprising theself-assembling polypeptide may additionally comprise one or morepharmaceutical compounds.

The self-assembling polypeptide, as described herein, may also beapplied to several preferential objects including, but not limited to,meshes, scaffolds, patches, nonwovens, and implants, for example, dentalimplants, microchip implants, soft tissue implants such as siliconeimplants, or implants with a silicone surface (e.g. cochlea implants).Due to this application, the objects including, but not limited to,meshes, scaffolds, patches, nonwovens, and implants, for example, dentalimplants, microchip implants, soft tissue implants such as siliconeimplants, or implants with a silicone surface (e.g. cochlea implants)may be covered or coated, e.g. partially or completely covered orcoated, with the self-assembling polypeptide.

It can be applied to these objects via dip-coating, spraying, ordropping. For dip-coating, preferential objects are dipped into liquidcompositions such as aqueous compositions, e.g. aqueous solutions,comprising the self-assembling polypeptide. Further, for spraying,liquid compositions such as aqueous compositions, e.g. aqueoussolutions, comprising the self-assembling polypeptide are sprayed ontopreferential objects. Furthermore, for dropping, liquid compositionssuch as aqueous compositions, e.g. aqueous solutions, comprising theself-assembling polypeptide are dropped onto preferential objects. Theseliquid compositions comprising the self-assembling polypeptide mayadditionally comprise one or more pharmaceutical compounds such astherapeutic or diagnostic compounds.

The term “pharmaceutical compound”, as used herein, is defined below.Preferably, the pharmaceutical compound is selected from the groupconsisting of an anti-microbial compound, an anti-viral compound, ananti-fungal compound, an immunosuppressive compound, a growth factor, anenzyme, an anti-inflammatory compound, an anti-allergic compound, asedative compound, a protein, particularly a glycoprotein orlipoprotein, a polysaccharide, and mixtures thereof.

Thus, in a preferred embodiment of the present invention, theself-assembling polypeptide, as described herein, is for use as tissueadhesive, wherein the self-assembling polypeptide is provided (i) asfilm, gel, particularly hydrogel, foam, mesh, scaffold, patch, nonwovenor layer, particularly covering or coating layer, or (ii) comprised inand/or on an object, e.g. a mesh, scaffold, patch, nonwoven or implant,for example, dental implant, microchip implant, soft tissue implant suchas silicone implant or implant with a silicone surface (e.g. cochleaimplant). Said object (e.g. mesh, scaffold, patch or nonwoven), may beinserted into the wound (e.g. inserted into the cut or gap of the wound)and may be left in the wound.

In preferred embodiments of the invention, the self-assemblingpolypeptide, as described herein, is for use as tissue adhesive

-   -   i) in the treatment of a wound,    -   ii) in the treatment of a sutured wound,    -   iii) in the reduction or prevention of fibrosis, particularly        capsular fibrosis, and/or in the reduction or prevention of        scarring,    -   iv) in the fixation of transplants, preferably tissue        transplants or organ transplants, more preferably skin        transplants,    -   v) in the fixation of medical devices, preferably implants, more        preferably silicone implants or implants with a silicone        surface, and/or    -   vi) in surgical interventions.

As mentioned above, the self-assembling polypeptide, as describedherein, is for use as tissue adhesive in the treatment of a wound. Theinventors of the present invention surprisingly found that theself-assembling polypeptide, as described herein, can be used toreconnect tissue layers of a wound with each other. Particularly, thetissue adhesive can provide a close, especially form-fit, connectionbetween tissue layers, or in the event that the tissue layers aredistant from each other, the tissue adhesive can fill the gap betweenthe tissue layers, replace the missing tissue layers and/or bridge themissing tissue layers. Preferably, the gap has a size of no more than 1cm, more preferably of no more than 0.75 cm, even more preferably of nomore than 0.5 cm, most preferably of no more than 0.25 cm, e.g. of nomore than 0.01, 0.015, 0.02, 0.025, 0.0.3, 0.035, 0.04, 0.045, 0.05,0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.15,0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8,0.85, 0.9, 0.95, or 1 cm.

The term “wound”, as used herein, includes damages to any tissue in apatient. As used herein, the term “patient” means any mammal or birdthat may benefit from the tissue adhesive as described herein.Preferably, the patient is selected from the group consisting of alaboratory animal (including, for example, a mouse or rat), a domesticanimal (including, for example, a guinea pig, rabbit, chicken, turkey,pig, sheep, goat, camel, cow, horse, donkey, cat, or dog), and primates(including, for example, a chimpanzee and a human being). It isparticularly preferred that the patient is a human being. Alternatively,the patient is selected from the group consisting of a human being andan animal.

The wound may be comprised on the surface of the body of a patient, e.g.human being, (i.e. a superficial wound), or may be comprised within thebody of a patient, e.g. human being (i.e. an internal wound). The woundmay have been caused by any means including, but not limited to,infections, inflammations, surgical interventions, external componentssuch as sharp objects, e.g. scalpels, knifes or nails, and externalcircumstances, such as accidents, e.g. bicycle, motor vehicle, or autoaccidents.

It is particularly preferred that the wound is selected from the groupconsisting of a topical wound, deep wound, gaping wound, stab wound,puncture wound, penetration wound, surgical incision, laceration, cut,and trauma (e.g. blunt or sharp trauma). The term “topical wound”, asused herein, refers to a wound on the tissue surface. The term “puncturewound”, as used herein, refers to a wound caused by an object puncturingtissue(s), e.g. multiple (different) tissues, particularly an organ,e.g. skin, such as a nail or needle. The term “penetration wound”, asused herein, refers to a wound caused by an object entering and comingout from the tissue(s), e.g. multiple (different) tissues, particularlyan organ, e.g. skin, such as a nail or needle. The term “surgicalincision”, as used herein, refers to a wound caused by a sharp object,e.g. a scalpel or knife, during surgery. The term “trauma”, as usedherein, is defined as an injury caused by a physical fore, e.g. includethe consequences of motor vehicle accidents.

The wound may be located in or on the surface of a tissue. The tissuemay be selected form the group consisting of connective tissue, muscletissue, nervous tissue, epithelial tissue, and combinations thereof,e.g. multiple (different) tissues. An organ, e.g. stomach, smallintestine, large intestine, bowel, rectum, oesophagus, lung, spleen,brain, heart, kidney, liver, skin, glands such as lymph and thyroidglands, eye, or pancreas, is, for example, comprised of multiple(different) tissues. Thus, the wound may also be located in an organ,particularly encompassing multiple (different) tissues or tissue layers.Particularly, the wound is a skin lesion.

In preferred embodiments of the invention, the self-assemblingpolypeptide, as described herein, is for use as tissue adhesive in (i)the topical treatment of a wound site and/or (ii) the treatment of aninternal wound site, e.g. in case of deeper wounds or during surgicalprocedures.

As mentioned above, the self-assembling polypeptide, as describedherein, is for use as tissue adhesive in the treatment of a suturedwound. The inventors of the present invention surprisingly found thatthe self-assembling polypeptide, as described herein, can be used tofurther seal a sutured wound. In this case, the self-assemblingpolypeptide acting as a tissue adhesive can further fix and/or glue thesutured tissue layers with each other. This may strengthen the effect ofthe wound suture, prevent after-bleeding, and/or avoid subsequentinfections, e.g. bacterial or viral infections. Sutured wounds which aretreated with the self-assembling polypeptide, as described herein,advantageously exhibit reduced or no scarring and/or show a shortenedhealing. It is particularly preferred that the sutured wound is selectedfrom the group consisting of a topical wound, deep wound, gaping wound,stab wound, puncture wound, penetration wound, surgical incision,laceration, cut, and trauma (e.g. blunt trauma or sharp trauma).

As mentioned above, the self-assembling polypeptide, as describedherein, is for use as tissue adhesive in the reduction or prevention offibrosis, particularly capsular fibrosis, and/or in the reduction orprevention of scarring. Preferably, wounds which are glued with theself-assembling polypeptide, particularly silk polypeptide, as describedherein, exhibit reduced or no scarring and/or show a reduced or nofibrosis, particularly capsular fibrosis.

Further, as mentioned above, the self-assembling polypeptide, asdescribed herein, is for use as tissue adhesive in the fixation oftransplants, preferably tissue transplants or organ transplants, morepreferably skin transplants. The inventors surprisingly found that theself-assembling polypeptide can be used in the fixation of transplants,preferably tissue transplants or organ transplants, more preferably skintransplants. Particularly skin transplants have the disadvantage thatthey tend to slip after application. This is especially caused bypatient movements which cannot be completely obviated. Under thesecircumstances, the correct healing process is delayed or even prevented.The skin transplants are preferably selected from the group consistingof autologous skin transplants (also designated as autografts),isogeneic skin transplants (also designated as isografts or syngrafts),allogeneic skin transplants (also designated as allografts), xenogenicskin transplants (also designated as xenografts or heterografts), andprosthetic skin transplants (also designated as prosthetic grafts). Themeaning of these terms is clear for the skilled person. In order to fixthe transplants, e.g. tissue transplants or organ transplants such asskin transplants, to tissue or in the body cavity, the surface area ofthe transplants, e.g. tissue transplants or organ transplants such asskin transplants, that will be in contact with the tissue or body cavitymay be covered or coated, e.g. partially or completely covered orcoated, with the self-assembling polypeptide. Alternatively oradditionally, the tissue or body cavity that will be in contact with thetransplants, e.g. tissue transplants or organs transplants such as skintransplants, may be covered or coated, e.g. partially or completelycovered or coated, with the self-assembling polypeptide.

Furthermore, as mentioned above, the self-assembling polypeptide, asdescribed herein, is for use as a tissue adhesive in the fixation ofmedical devices such as implants. The inventors surprisingly found thatthe self-assembling polypeptide, as described herein, can be used toconnect tissue layers to artificial surfaces, e.g. of medical devicessuch as implants, or can be used to connect artificial surfaces, e.g. ofmedical devices such as implants, to tissue layers. For this purpose,the tissue layers or artificial surfaces, e.g. of the medical devicessuch as implants, can be covered or coated, e.g. partially or completelycovered or coated, with the self-assembling polypeptide. Thereby, themedical devices such as implants are fixed, particularly embedded orincorporated, in the tissue.

The term “medical device”, as used herein, refers to an instrument,apparatus, implant, or other similar or related article, which isintended for use in the diagnosis of diseases or other conditions, or inthe cure, mitigation, treatment or prevention of diseases, or which isintended to affect the structure or any function of the body and whichdoes not achieve any of its primary intended purposes through chemicalaction within or on the body. Particularly, medical devices achievetheir principal action by physical, physico-chemical, or mechanicalmeans. It is preferred that the medical devices are selected from thegroup consisting of implants, preferably dental implants, microchipimplants, soft tissue implants such as silicone implants, or implantswith a silicone surface (e.g. cochlea implants), cardiac pacemakers,pumps, preferably insulin pumps, cannula, preferably cannula for thedrainage of liquor, ichor, or blood, deposits, preferably drug deposits,fixations, preferably internal or external fixations (“fixateur externe”in French), prostheses, preferably neuroprostheses, and sensors,preferably sensors measuring the body temperature, blood pressure, orpulse rate. The silicone implants or implants with a silicone surfacemay also be breast implants.

The medical device may be a subdermal or transdermal medical device,e.g. a transdermal or subdermal implant. A subdermal medical device is adevice that is completely buried in the skin, while a transdermalmedical device is placed under the skin, but also protrudes out of it.

In addition, as mentioned above, the self-assembling polypeptide asdescribed herein is for use in surgical interventions. It is alsoparticularly preferred that the surgical interventions are selected fromthe group consisting of cardiovascular surgical interventions,cardiothoracic surgical interventions, gastrointestinal surgicalinterventions, pneumothoracic surgical interventions, neurosurgicalinterventions, urological surgical interventions, dental surgicalinterventions, reconstructive surgical interventions, surgicalinterventions in the ear, surgical interventions in the nose, surgicalinterventions in the throat area, lymphatic, biliary and cerebrospinalfistulae, air leakages during thoracic and pulmonary surgicalinterventions, orthopaedic surgical interventions, gynaecologicalsurgical interventions, cosmetical surgical interventions, and vascularsurgical interventions. It is particularly preferred that the cosmeticalor reconstructive surgical interventions include the insertion ofimplants, preferably dental implants, soft tissue implants such assilicone implants, or implants with a silicone surface (e.g. cochleaimplants). The silicone implants or implants with a silicone surface mayalso be breast implants.

The self-assembling polypeptide as tissue adhesive may be used as partof a composition such as an aqueous solution having a temperature of 20°C. and a pH of between 4.5 and 7.0 such as of between 4.5 and 6.5,between 4.8 and 5.9, or of between 5.4 and 5.9. The adhesive effect ofthe self-assembling polypeptide can be improved by increasing thetemperature of the composition such as aqueous solution above 20° C.,e.g. from a temperature of 20° C. to 25° C., 30° C., 35° C., 37° C., 38°C., 39° C. or 40° C. Alternatively or additionally, the adhesive effectof the self-assembling polypeptide can be improved by decreasing the pHof the composition such as aqueous solution by 0.1 to 2.5 pH units, e.g.by 0.1 to 0.5 pH units, below the pH of the tissue to be glued. Thetissue to be glued may have a pH of between 4.5 and 7.0 such as ofbetween 4.5 and 6.5, of between 4.8 and 5.9, or of between 5.4 and 5.9.

The first aspect of the present invention, as described above, canalternatively be worded as follows: In a first aspect, the presentinvention relates to a method of using a self-assembling polypeptide astissue adhesive.

In a second aspect, the present invention relates to the use of aself-assembling polypeptide as tissue adhesive. Said use may be an invivo, in vitro or ex vivo use, preferably an in vitro or ex vivo use. Asto the definition of the terms “self-assembling polypeptide” and “tissueadhesive”, it is referred to the first aspect of the present invention.The tissue is preferably selected from the group consisting ofconnective tissue, muscle tissue, nervous tissue, epithelial tissue, andcombinations thereof, e.g. multiple (different) tissues. An organ, e.g.stomach, small intestine, large intestine, bowel, rectum, oesophagus,lung, spleen, brain, heart, kidney, liver, skin, adrenal glands, lymphand thyroid glands, eye, or pancreas, is, for example, comprised ofmultiple (different) tissues. As to the preferred embodiments of the“self-assembling polypeptide”, particularly silk polypeptide, elastin,collagen, or keratin, it is also referred to the first aspect of thepresent invention. For example, it is preferred that the self-assemblingpolypeptide further comprises at least one peptide which is capable ofenhancing the adhesive effect. It is further preferred that the adhesiveeffect is mediated via binding of the peptide to a cell adhesionmediating protein (CAMP). It is also, additionally or alternatively,preferred that the peptide comprises at least one CAMP recognitionsequence. As to the definition of the terms “peptide”, “peptide which iscapable of enhancing the adhesive effect”, “cell adhesion mediatingprotein (CAMP)”, and “cell adhesion” and as to preferred embodiments ofthe “peptide”, “peptide which is capable of enhancing the adhesiveeffect”, “cell adhesion mediating protein”, “CAMP recognition sequence”,it is referred to the first aspect of the present invention.

As mentioned above, the present invention relates to the use of aself-assembling polypeptide, e.g. silk polypeptide such as spider silkpolypeptide, as tissue adhesive. Preferably, the self-assemblingpolypeptide is used to process food. In one embodiment, theself-assembling polypeptide is used to glue tissues, preferably meat,more preferably pork, beef, chicken, or turkey.

The second aspect of the present invention, as described above, canalternatively be worded as follows: In a second aspect, the presentinvention relates to a method of using a self-assembling polypeptide astissue adhesive.

In a third aspect, the present invention relates to the use of aself-assembling polypeptide to glue one or more cosmetic compounds onskin, mucosa, and/or hair, particularly on skin surfaces, mucosasurfaces and/or hair surfaces.

As to the definition of the term “self-assembling polypeptide” and as topreferred embodiments of the “self-assembling polypeptide”, particularlysilk polypeptide, elastin, collagen, or keratin, it is referred to thefirst aspect of the present invention. For example, it is preferred thatthe self-assembling polypeptide further comprises at least one peptidewhich is capable of enhancing the adhesive effect. It is furtherpreferred that the adhesive effect is mediated via binding of thepeptide to a cell adhesion mediating protein (CAMP). It is also,additionally or alternatively, preferred that the peptide comprises atleast one CAMP recognition sequence. As to the definition of the terms“peptide”, “peptide which is capable of enhancing the adhesive effect”,“cell adhesion mediating protein (CAMP)”, and “cell adhesion” and as topreferred embodiments of the “peptide”, “peptide which is capable ofenhancing the adhesive effect”, “cell adhesion mediating protein”, “CAMPrecognition sequence”, it is referred to the first aspect of the presentinvention.

The term “cosmetic compound (also designated as cosmetic substance)”, asused herein, refers to a substance intended mainly for external use onthe body surface, e.g. human body surface, or in the oral cavity, e.g.of a human, for cleaning and personal hygiene to alter the appearance orbody odor or to convey scent. In particular, it is meant that a cosmeticsubstance is a molecule which shows a certain predictable effect. Suchan effect molecule can be, for example, a proteinaceous molecule (e.g.an enzyme) or a non-proteinaceous molecule (e.g. a dye, pigment,photo-protective agent, vitamin, provitamin, an antioxidant,conditioner, or a compound comprising metal ions).

Among the proteinaceous molecules, enzymes are preferred. Examples foruseful enzymes include, but are not limited to, oxidases, peroxidases,proteases, glucanases, mutanases, tyrosinases, metal-binding enzymes,lactoperoxidases, lysozymes, aminoglycosidases, glucose oxidases, superoxide dismutases, photolyases, proteins binding heavy metals, T4endonucleases, catalases, and reductases such as thioredoxin-reductases.Also preferred are proteinaceous substances which do not possess anenzymatic function. Examples for non-enzymatic proteinaceous moleculesinclude, but are not limited to, antimicrobial peptides, hydrophobins,collagens, keratins, proteins binding heavy metals, proteins bindingodorants, proteins binding cellulose, proteins binding starch, andproteins binding keratin. Other preferred proteinaceous molecules are,for example, protein hydrolysates, e.g. protein hydrolysates of plant oranimal sources. Said protein hydrolysates can be of marine origin.

Among the non-proteinaceous molecules, UV-protective agents,antioxidants, vitamins, provitamins and their precursors and theirderivatives, dyes, polysaccharides, or fragrances are preferred. Theterm “UV-protective agent”, as used herein, refers to an organicsubstance which can absorb specific wavelengths in the range ofUV-wavelengths. The absorbed energy can then emitted in form of longerwave radiation, e.g. heat. The term “antioxidant”, as used herein,refers to a compound that interrupts the photochemical reaction chaintriggered by UV radiation when penetrating into the skin. Typicalexamples of antioxidants include, but are not limited to, super oxidedismutase, catalase, tocopherol (vitamin E), ascorbic acid (vitamin C),coenzyme Q10 (ubiquinane), and quinione. Examples of vitamins,provitamins and their precursors include, but are not limited to,β-carotene (provitamin of vitamin A), ascorbic acid (vitamin C),tocopherol (vitamin E), the vitamins, provitamins and their precursorsof the vitamin B group encompassing vitamin B₁ (thiamine), vitamin B₂(riboflavin), vitamin B3 (nicotinic acid or nicotinamid), vitamin B5(panthothenic acid and panthenol), vitamin B₆(5-hydroxymethyl-2-methylpyridin-3-ol, also known as pyridoxine,pyridoasamine or pyridoxal) and vitamin B7 (biotin). Examples of dyesinclude, but are not limited to, food dyes, semi-permanent dyes,permanent dyes, reactive dyes, and oxidation dyes. Useful dyes are forexample described in Rowe Colour Index, 3^(rd) edition, Society of Dyersand Colourists, Bradford, England, 1971. The use of the self-assemblingprotein particularly allows the gluing of tattoos such as temporarytattoos on skin surfaces.

In addition, the cosmetic compounds may be proteins, particularlyglycoproteins or lipoproteins, fragrances, stem cells,infrared-reflective compounds, infrared-absorbent compounds, mixtures ofinfrared-reflective compounds and infrared-absorbent compounds, arganoils, hyaluronic acids, sea silt extracts, gelee royale, gold extracts,medihoney, sacha inchi-oils, or allatonins.

More preferably, the cosmetic compounds are dyes, fragrances, stemcells, enzymes, vitamins, UV protective agents, or antioxidants.

As mentioned above, the self-assembling polypeptide is used to glue oneor more cosmetic compounds on skin, mucosa, and/or hair. The skin may beselected from the group consisting of facial skin, the skin of the arms(e.g. upper or under arms), the skin of the feet, and the skin of thelegs, the mucosa may be selected from the group consisting of oralmucosa, nasal mucosa, lingual mucosa, labial mucosa, and palatal mucosa,and/or the hair may be selected from the group consisting of scalp hair,eyelashes, the hair of the arms, the hair of the feet, the hair of thelegs, facial hair, and pubic hair.

The self-assembling polypeptide may be used as part of a compositionsuch as an aqueous solution having a temperature of 20° C. and a pH ofbetween 4.5 and 7.0 such as of between 4.5 and 6.5, between 4.8 and 5.9,or of between 5.4 and 5.9 in order to glue one or more cosmeticcompounds on skin, mucosa, and/or hair. The adhesive effect of theself-assembling polypeptide can be improved by increasing thetemperature of the composition such as aqueous solution above 20° C.,e.g. from a temperature of 20° C. to 25° C., 30° C., 35° C., 37° C., 38°C., 39° C. or 40° C. Alternatively or additionally, the adhesive effectof the self-assembling polypeptide can be improved by decreasing the pHof the composition such as aqueous solution by 0.1 to 2.5 pH units, e.g.by 0.1 to 0.5 pH units, below the pH of the skin, mucosa, and/or hair tobe treated. The skin, mucosa, and/or hair to be treated may have a pH ofbetween 4.5 and 7.0 such as of between 4.5 and 6.5, of between 4.8 and5.9, or of between 5.4 and 5.9.

The third aspect of the present invention, as described above, canalternatively be worded as follows: In a third aspect, the presentinvention relates to a method of using a self-assembling polypeptide toglue one or more cosmetic compounds on skin, mucosa, and/or hair.

In a fourth aspect, the present invention relates to a self assemblingpolypeptide for use in gluing one or more pharmaceutical compounds ontissue, skin, mucosa, and/or hair.

As to the definition of the term “self-assembling polypeptide” and as topreferred embodiments of the “self-assembling polypeptide”, particularlysilk polypeptide, elastin, collagen, or keratin, it is referred to thefirst aspect of the present invention. For example, it is preferred thatthe self-assembling polypeptide further comprises at least one peptidewhich is capable of enhancing the adhesive effect. It is furtherpreferred that the adhesive effect is mediated via binding of thepeptide to a cell adhesion mediating protein (CAMP). It is also,additionally or alternatively, preferred that the peptide comprises atleast one CAMP recognition sequence. As to the definition of the terms“peptide”, “peptide which is capable of enhancing the adhesive effect”,“cell adhesion mediating protein (CAMP)”, and “cell adhesion” and as topreferred embodiments of the “peptide”, “peptide which is capable ofenhancing the adhesive effect”, “cell adhesion mediating protein”, “CAMPrecognition sequence”, it is referred to the first aspect of the presentinvention.

The term “pharmaceutical compound”, as used herein, refers to anybiological or chemical substance, particularly pharmacological,metabolic, or immunological substance, which may be used in thetreatment, cure, prophylaxis, prevention, or diagnosis of a pathologicalcondition, e.g. a disease or disorder, or which may be used to otherwiseenhance physical, psychical or mental well-being. Accordingly, the term“pharmaceutical compound” envisaged in the context of the presentinvention includes any compound with therapeutic, diagnostic, orprophylactic effects. For example, the pharmaceutical compound can be acompound that affects or participates in tissue growth, cell growth,cell differentiation, a compound that is able to invoke a biologicalaction such as an immune response, or a compound that can play any otherrole in one or more biological processes. Preferably, the pharmaceuticalcompound is selected from the group consisting of an anti-microbialcompound, such as an antibacterial compound (e.g. an antibiotic), ananti-viral compound or an anti-fungal compound, an immunosuppressivecompound, an anti-inflammatory compound, an anti-allergic compound, ananti-coagulant, an anti-rheumatic compound, an anti-psoriatic compound,a sedative compound, a muscle relaxant, an anti-migraine compound, ananti-depressant, an insect repellent, a growth factor, a hormone, ahormone antagonist, an antioxidant, a protein, such as a glycoprotein,lipoprotein, or an enzyme (e.g. hyaluronidases), a polysaccharide, afree radical scavenger, a radio-therapeutic compound, a photodynamictherapy compound, a dye such as a fluorescent dye, and a contrast agent.

As mentioned above, the self assembling polypeptide is for use in gluingone or more pharmaceutical compounds on tissue, skin, mucosa, and/orhair. The tissue may be selected from the group consisting of connectivetissue, muscle tissue, nervous tissue, epithelial tissue, andcombinations thereof (e.g. multiple (different) tissues such as from anorgan, for example, stomach, small intestine, large intestine, bowel,rectum, oesophagus, lung, spleen, brain, heart, kidney, liver, skin,adrenal glands, lymph and thyroid glands, eye, or pancreas), the skinmay be selected from the group consisting of facial skin, the skin ofthe arms (e.g. upper or under arms), the skin of the feet, and the skinof the legs, the mucosa may be selected from the group consisting oforal mucosa, nasal mucosa, lingual mucosa, labial mucosa, palatalmucosa, alveolar mucosa, bowel mucosa, bronchial mucosa, gastric mucosa,intestinal mucosa, ruminal mucosa and stomach mucosa, and/or the hairmay be selected from the group consisting of scalp hair, eyelashes, thehair of the arms, the hair of the feet, the hair of the legs, facialhair, and pubic hair. Particularly, the tissue may be a peri-operativeor post-operative tissue.

The self-assembling polypeptide may be used as part of a compositionsuch as an aqueous solution having a temperature of 20° C. and a pH ofbetween 4.5 and 7.0 such as of between 4.5 and 6.5, between 4.8 and 5.9,or of between 5.4 and 5.9 in order to glue one or more pharmaceuticalcompounds on tissue, skin, mucosa, and/or hair. The adhesive effect ofthe self-assembling polypeptide can be improved by increasing thetemperature of the composition such as aqueous solution above 20° C.,e.g. from a temperature of 20° C. to 25° C., 30° C., 35° C., 37° C., 38°C., 39° C. or 40° C. Alternatively or additionally, the adhesive effectof the self-assembling polypeptide can be improved by decreasing the pHof the composition such as aqueous solution by 0.1 to 2.5 pH units, e.g.by 0.1 to 0.5 pH units, below the pH of the tissue, skin, mucosa, and/orhair to be treated. The tissue, skin, mucosa, and/or hair to be treatedmay have a pH of between 4.5 and 7.0 such as of between 4.5 and 6.5, ofbetween 4.8 and 5.9, or of between 5.4 and 5.9.

The fourth aspect of the present invention, as described above, canalternatively be worded as follows: In a fourth aspect, the presentinvention relates to a method of using a self-assembling polypeptide toglue one or more pharmaceutical compounds on tissue, skin, mucosa,and/or hair.

In a fifth aspect, the present invention relates to an applicationcombination (or simply to a combination) comprising

-   i) a self-assembling polypeptide, and-   ii) a factor enhancing self-assembly    for use as tissue adhesive.

The term “application combination”, as used herein, refers to acombination of at least two components, a self-assembling polypeptideand a factor enhancing self-assembly, which can be applied to surfacesto be glued. The application combination may be applied to tissue tofunction as tissue adhesive, particularly to (re)connect tissue layerswith each other (see fifth and sixth aspect of the present invention).It may also be applied to skin, mucosa, and/or hair to glue one or morecosmetic compounds to these surfaces (see seventh aspect of the presentinvention) or to tissue, skin, mucosa, and/or hair to glue one or morepharmaceutical compounds to these surfaces (see eighth aspect of thepresent invention).

As to the definition of the terms “self-assembling polypeptide”, “selfassembly”, and “tissue adhesive”, it is referred to the first aspect ofthe present invention. The tissue is preferably selected from the groupconsisting of connective tissue, muscle tissue, nervous tissue,epithelial tissue, and combinations thereof, e.g. multiple (different)tissues. An organ, e.g. stomach, small intestine, large intestine,bowel, rectum, oesophagus, lung, spleen, brain, heart, kidney, liver,skin, adrenal glands, lymph and thyroid glands, eye, or pancreas, is,for example, comprised of multiple (different) tissues. As to thepreferred embodiments of the “self-assembling polypeptide”, particularlysilk polypeptide, elastin, collagen, or keratin, it is also referred tothe first aspect of the present invention. For example, it is preferredthat the self-assembling polypeptide further comprises at least onepeptide which is capable of enhancing the adhesive effect. It is furtherpreferred that the adhesive effect is mediated via binding of thepeptide to a cell adhesion mediating protein (CAMP). It is also,additionally or alternatively, preferred that the peptide comprises atleast one CAMP recognition sequence. As to the definition of the terms“peptide”, “peptide which is capable of enhancing the adhesive effect”,“cell adhesion mediating protein (CAMP)”, and “cell adhesion” and as topreferred embodiments of the “peptide”, “peptide which is capable ofenhancing the adhesive effect”, “cell adhesion mediating protein”, “CAMPrecognition sequence”, it is referred to the first aspect of the presentinvention.

The term “a factor enhancing self-assembly”, as used herein, refers to amolecule which enhances the process in which a disordered system ofpre-existing polypeptides forms an organised structure or pattern as aconsequence of specific, local interactions (e.g. van der Waals forces,hydrophobic interactions, hydrogen bonds, and/or salt-bridges, etc.)among the polypeptides themselves. It particularly enhances this processby accelerating self-assembly. The change from a disordered system to anorganised structure or pattern during self-assembly is characterized bya transition from a fluid state to a gelatinous and/or solid state and acorresponding increase in viscosity. The transition from a fluid to agelatinous state can be monitored, for example, by measurement of lightscattering, rheology, or Circular Dichroism (CD). The transition from afluid to a solid state can be monitored, for example, by opticalmethods. These techniques are known to the skilled person. Preferably,the factor enhancing self-assembly is selected from the group consistingof alcohols, sulfates, phosphates, and a cross-linking agent.

The term “cross-linking” agent refers to a compound which is able toform chemical links between molecular chains such as protein chains tobuild a three-dimensional network of connected molecules such asproteins. Preferably, the cross-linking agent has the following formula(I):

whereinR1 is selected from the group consisting of H, C₁ to C₆ alkyl,preferably C₁ to C₄ alkyl, C₁ to C₆ hydroxyalkyl, preferably C₁ to C₄hydroxyalkyl, and CH₂—C(O)O—R4, wherein R4 is C₁ to C₄ alkyl,R2 is selected from the group consisting of H, C₁ to C₆ alkyl,preferably C₁ to C₄ alkyl, C₁ to C₆ hydroxyalkyl, preferably C₁ to C₄hydroxyalkyl, and CH₂—C(O)O—R4, wherein R4 is C₁ to C₄ alkyl, andR3 is selected from the group consisting of H, C₁ to C₆ alkyl,preferably C₁ to C₄ alkyl, more preferably CH₃, and C₁ to C₆hydroxyalkyl, preferably C₁ to C₄ hydroxyalkyl.It is particularly preferred that R1 and R2 are identical, e.g. that R1and R2 are H, and/or that R3 is H or CH₃. More preferably, thecross-linking agent is genipin having the following formula (II):

Genipin is a chemical compound found in gardenia fruit extract. It is anaglycone derived from an iridoid glycoside called geniposide present infruit of Gardenia jasminoides. Genipin is an excellent naturalcross-linker for proteins. It enhances or facilitates self-assembly ofpolypeptides such as silk polypeptides.

It is preferred that the self-assembling polypeptide and the factorenhancing self-assembly are consecutively or simultaneously applied. Forconsecutive applications, the self-assembling polypeptide may be appliedfirst and the factor enhancing self-assembly may be applied afterwards,or the factor enhancing self-assembly may be applied first and theself-assembling polypeptide may be applied afterwards. For example, thefirst component (e.g. the self-assembling polypeptide or the factorenhancing self-assembly) is spread on the tissue area, e.g. wound, andthen the second component (e.g. the factor enhancing self-assembly orthe self-assembling polypeptide) is spread on the same tissue area, e.g.wound. For simultaneous applications, the self-assembling polypeptideand the factor enhancing self-assembly may be applied at the same time,preferably comprised in a two component application combination deviceor in form of a two component application combination. For example, thetwo components (i.e. the self-assembling polypeptide and the factorenhancing self-assembly) of the two component application combinationdevice are spread simultaneously on the tissue area, e.g. wound. The twocomponent application combination device may include two separatesyringes, one may be filled with the self-assembling polypeptide and onemay be filled with the factor enhancing self-assembly. A dual syringetwo component application combination device may also be used.

It is further preferred that

-   -   i) the self-assembling polypeptide is comprised in a        composition, and/or    -   ii) the factor enhancing self-assembly is comprised in a        composition.

The composition may be a solid composition such as a powderycomposition, a liquid composition such as an aqueous composition, e.g.an aqueous solution, an emulsion, or a suspension, an aerosol such as adry or liquid aerosol, a pump spray, or a pasty composition. Preferably,the composition comprising the self-assembling polypeptide and thecomposition comprising the factor enhancing self-assembly are of thesame type. More preferably, the self-assembling polypeptide is comprisedin a liquid composition, particularly in an aqueous composition, e.g. anaqueous solution, and/or the factor enhancing self-assembly is comprisedin a liquid composition, particularly aqueous composition, e.g. aqueoussolution.

It is particularly preferred that the composition comprising theself-assembling polypeptide and/or the composition comprising the factorenhancing self-assembly further comprises one or more pharmaceuticalcompounds. The term “pharmaceutical compound” is defined above.Preferably, the pharmaceutical compound is selected from the groupconsisting of an anti-microbial compound, an anti-viral compound, ananti-fungal compound, an immunosuppressive compound, a growth factor, anenzyme, an anti-inflammatory compound, an anti-allergic compound, asedative compound, a protein, particularly a glycoprotein orlipoprotein, a polysaccharide, and mixtures thereof.

In preferred embodiments of the invention, the application compositioncomprising the self-assembling polypeptide and the factor enhancingself-assembly, as described herein, is for use as tissue adhesive

-   -   i) in the treatment of a wound,    -   ii) in the treatment of a sutured wound,    -   iii) in the reduction or prevention of fibrosis, particularly        capsular fibrosis, and/or in the reduction or prevention of        scarring,    -   iv) in the fixation of transplants, preferably tissue        transplants or organ transplants, more preferably skin        transplants,    -   v) in the fixation of medical devices, preferably implants, more        preferably silicone implants or implants with a silicone        surface, and/or    -   vi) in surgical interventions.

As to the definition of the terms “wound” and “medical devices” and asto the preferred embodiments of the “wounds”, “sutured wounds”,“transplants”, “medical devices”, “implants”, and “surgicalinterventions”, it is referred to the first aspect of the presentinvention.

The fifth aspect of the present invention, as described above, canalternatively be worded as follows: In a fifth aspect, the presentinvention relates to a method of using an application combinationcomprising a self-assembling polypeptide and a factor enhancingself-assembly as tissue adhesive.

In a sixth aspect, the present invention relates to the use of anapplication combination (or simply of a combination) comprising

-   -   i) a self-assembling polypeptide, and    -   ii) a factor enhancing self-assembly as tissue adhesive.

The use may be an in vivo, in vitro, or ex vivo use, preferably an invitro or ex vivo use. As to the definition of the terms “self-assemblingpolypeptide”, “self assembly”, and “tissue adhesive”, it is referred tothe first aspect of the present invention. The tissue is preferablyselected from the group consisting of connective tissue, muscle tissue,nervous tissue, epithelial tissue, and combinations thereof, e.g.multiple (different) tissues. An organ, e.g. stomach, small intestine,large intestine, bowel, rectum, oesophagus, lung, spleen, brain, heart,kidney, liver, skin, adrenal glands, lymph and thyroid glands, eye, orpancreas, is, for example, comprised of multiple (different) tissues. Asto the preferred embodiments of the “self-assembling polypeptide”,particularly “silk polypeptide”, it is also referred to the first aspectof the present invention. For example, it is preferred that theself-assembling polypeptide further comprises at least one peptide whichis capable of enhancing the adhesive effect. It is further preferredthat the adhesive effect is mediated via binding of the peptide to acell adhesion mediating protein (CAMP). It is also, additionally oralternatively, preferred that the peptide comprises at least one CAMPrecognition sequence. As to the definition of the terms “peptide”,“peptide which is capable of enhancing the adhesive effect”, “celladhesion mediating protein (CAMP)”, and “cell adhesion” and as topreferred embodiments of the “peptide”, “peptide which is capable ofenhancing the adhesive effect”, “cell adhesion mediating protein”, “CAMPrecognition sequence”, it is referred to the first aspect of the presentinvention. Further, as to the definition of the terms “applicationcombination”, and “a factor enhancing self-assembly”, it is referred tothe fifth aspect of the present invention. Preferably, the factorenhancing self-assembly is selected from the group consisting ofalcohols, sulfates, phosphates, and a cross-linking agent. As to thedefinition of the term “cross-linking agent” and as to preferredembodiments of the “cross-linking agent”, it is referred to the fifthaspect of the present invention. It is particularly preferred that thecross-linking agent is genipin.

It is preferred that the self-assembling polypeptide and the factorenhancing self-assembly are consecutively or simultaneously applied. Forconsecutive applications, the self-assembling polypeptide may be appliedfirst and the factor enhancing self-assembly may be applied afterwards,or the factor enhancing self-assembly may be applied first and theself-assembling polypeptide may be applied afterwards. For simultaneousapplications, the self-assembling polypeptide and the factor enhancingself-assembly may be applied at the same time, preferably comprised in atwo component application combination device or in form of a twocomponent application combination. For example, the two components (i.e.the self-assembling polypeptide and the factor enhancing self-assembly)of the two component application combination device are spreadsimultaneously on the tissue area, e.g. wound. The two componentapplication combination device may include two separate syringes, onemay be filled with the self-assembling polypeptide and one may be filledwith the factor enhancing self-assembly. A dual syringe two componentapplication combination device may also be used.

It is further preferred that

-   -   i) the self-assembling polypeptide is comprised in a        composition, and/or    -   ii) the factor enhancing self-assembly is comprised in a        composition.

The composition may be a solid composition such as a powderycomposition, a liquid composition such as an aqueous composition, e.g.an aqueous solution, an emulsion, or a suspension, an aerosol such as adry or liquid aerosol, a pump spray, or a pasty composition. Preferably,the composition comprising the self-assembling polypeptide and thecomposition comprising the factor enhancing self-assembly are of thesame type. More preferably, the self-assembling polypeptide is comprisedin a liquid composition, particularly in an aqueous composition, e.g. anaqueous solution, and/or the factor enhancing self-assembly is comprisedin a liquid composition, particularly aqueous composition, e.g. aqueoussolution.

It is particularly preferred that the composition comprising theself-assembling polypeptide and/or the composition comprising the factorenhancing self-assembly further comprises one or more pharmaceuticalcompounds. The term “pharmaceutical compound” is defined above.Preferably, the pharmaceutical compound is selected from the groupconsisting of an anti-microbial compound, an anti-viral compound, ananti-fungal compound, an immunosuppressive compound, a growth factor, anenzyme, an anti-inflammatory compound, an anti-allergic compound, asedative compound, a protein, particularly a glycoprotein orlipoprotein, a polysaccharide, and mixtures thereof.

As mentioned above, the present invention relates to the use of anapplication combination comprising a self-assembling polypeptide, e.g.silk polypeptide such as spider silk polypeptide, and a factor enhancingself-assembly as tissue adhesive. Preferably, the applicationcombination is used to process food. In one embodiment, the applicationcombination is used to glue tissues, preferably meat, more preferablypork, beef, chicken, or turkey.

The sixth aspect of the present invention, as described above, canalternatively be worded as follows: In a sixth aspect, the presentinvention relates to a method of using an application combinationcomprising a self-assembling polypeptide and a factor enhancingself-assembly as tissue adhesive.

In a seventh aspect, the present invention relates to the use of anapplication combination (or simply of a combination) comprising

-   -   i) a self-assembling polypeptide, and    -   ii) a factor enhancing self-assembly to glue one or more        cosmetic compounds on skin, mucosa, and/or hair.

As to the definition of the terms “self-assembling polypeptide” and“self-assembly” and as to preferred embodiments of the “self-assemblingpolypeptide”, particularly silk polypeptide, elastin, collagen, orkeratin, it is referred to the first aspect of the present invention.For example, it is preferred that the self-assembling polypeptidefurther comprises at least one peptide which is capable of enhancing theadhesive effect. It is further preferred that the adhesive effect ismediated via binding of the peptide to a cell adhesion mediating protein(CAMP). It is also, additionally or alternatively, preferred that thepeptide comprises at least one CAMP recognition sequence. As to thedefinition of the terms “peptide”, “peptide which is capable ofenhancing the adhesive effect”, “cell adhesion mediating protein(CAMP)”, and “cell adhesion” and as to preferred embodiments of the“peptide”, “peptide which is capable of enhancing the adhesive effect”,“cell adhesion mediating protein”, “CAMP recognition sequence”, it isreferred to the first aspect of the present invention. Further, as tothe definition of the terms “application combination”, and “a factorenhancing self-assembly”, it is referred to the fifth aspect of thepresent invention. Preferably, the factor enhancing self-assembly isselected from the group consisting of alcohols, sulfates, phosphates,and a cross-linking agent. As to the definition of the term“cross-linking agent” and as to preferred embodiments of the“cross-linking agent”, it is referred to the fifth aspect of the presentinvention. It is particularly preferred that the cross-linking agent isgenipin.

It is preferred that the self-assembling polypeptide and the factorenhancing self-assembly are consecutively or simultaneously applied. Forconsecutive applications, the self-assembling polypeptide may be appliedfirst and the factor enhancing self-assembly may be applied afterwards,or the factor enhancing self-assembly may be applied first and theself-assembling polypeptide may be applied afterwards. For example, thefirst component (e.g. the self-assembling polypeptide or the factorenhancing self-assembly) is spread on the tissue area, e.g. wound, andthen the second component (e.g. the factor enhancing self-assembly orthe self-assembling polypeptide) is spread on the same tissue area, e.g.wound. For simultaneous applications, the self-assembling polypeptideand the factor enhancing self-assembly may be applied at the same time,preferably comprised in a two component application combination deviceor in form of a two component application combination. For example, thetwo components (i.e. the self-assembling polypeptide and the factorenhancing self-assembly) of the two component application combinationdevice are spread simultaneously on the tissue area, e.g. wound. The twocomponent application combination device may include two separatesyringes, one may be filled with the self-assembling polypeptide and onemay be filled with the factor enhancing self-assembly. A dual syringetwo component application combination device may also be used.

It is further preferred that

-   -   i) the self-assembling polypeptide is comprised in a        composition, and/or    -   ii) the factor enhancing self-assembly is comprised in a        composition.

The composition may be a solid composition such as a powderycomposition, a liquid composition such as an aqueous composition, e.g.an aqueous solution, an emulsion, or a suspension, an aerosol such as adry or liquid aerosol, a pump spray, or a pasty composition. Preferably,the composition comprising the self-assembling polypeptide and thecomposition comprising the factor enhancing self-assembly are of thesame type. More preferably, the self-assembling polypeptide is comprisedin a liquid composition, particularly in an aqueous composition, e.g. anaqueous solution, and/or the factor enhancing self-assembly is comprisedin a liquid composition, particularly aqueous composition, e.g. aqueoussolution.

As to the definition of the term “cosmetic compound” and as to preferredembodiments of the “cosmetic compound”, “skin”, “mucosa”, and “hair”, itis referred to the third aspect of the present invention.

The seventh aspect of the present invention, as described above, canalternatively be worded as follows: In a seventh aspect, the presentinvention relates to a method of using an application combinationcomprising a self-assembling polypeptide and a factor enhancingself-assembly to glue one or more cosmetic compounds on skin, mucosa,and/or hair.

In an eighth aspect, the present invention relates to an applicationcombination (or simply a combination) comprising

-   -   i) a self-assembling polypeptide, and    -   ii) a factor enhancing self-assembly for use in gluing one or        more pharmaceutical compounds on tissue, skin, mucosa, and/or        hair.

As to the definition of the terms “self-assembling polypeptide” and“self-assembly” and as to preferred embodiments of the “self-assemblingpolypeptide”, particularly silk polypeptide, elastin, collagen, orkeratin, it is referred to the first aspect of the present invention.For example, it is preferred that the self-assembling polypeptidefurther comprises at least one peptide which is capable of enhancing theadhesive effect. It is further preferred that the adhesive effect ismediated via binding of the peptide to a cell adhesion mediating protein(CAMP). It is also, additionally or alternatively, preferred that thepeptide comprises at least one CAMP recognition sequence. As to thedefinition of the terms “peptide”, “peptide which is capable ofenhancing the adhesive effect”, “cell adhesion mediating protein(CAMP)”, and “cell adhesion” and as to preferred embodiments of the“peptide”, “peptide which is capable of enhancing the adhesive effect”,“cell adhesion mediating protein”, “CAMP recognition sequence”, it isreferred to the first aspect of the present invention. Further, as tothe definition of the terms “application combination”, and “a factorenhancing self-assembly”, it is referred to the fifth aspect of thepresent invention. Preferably, the factor enhancing self-assembly isselected from the group consisting of alcohols, sulfates, phosphates,and a cross-linking agent. As to the definition of the term“cross-linking agent” and as to preferred embodiments of the“cross-linking agent”, it is referred to the fifth aspect of the presentinvention. It is particularly preferred that the cross-linking agent isgenipin.

It is preferred that the self-assembling polypeptide and the factorenhancing self-assembly are consecutively or simultaneously applied. Forconsecutive applications, the self-assembling polypeptide may be appliedfirst and the factor enhancing self-assembly may be applied afterwards,or the factor enhancing self-assembly may be applied first and theself-assembling polypeptide may be applied afterwards. For example, thefirst component (e.g. the self-assembling polypeptide or the factorenhancing self-assembly) is spread on the tissue area, e.g. wound, andthen the second component (e.g. the factor enhancing self-assembly orthe self-assembling polypeptide) is spread on the same tissue area, e.g.wound. For simultaneous applications, the self-assembling polypeptideand the factor enhancing self-assembly may be applied at the same time,preferably comprised in a two component application combination deviceor in form of a two component application combination. For example, thetwo components (i.e. the self-assembling polypeptide and the factorenhancing self-assembly) of the two component application combinationdevice are spread simultaneously on the tissue area, e.g. wound. The twocomponent application combination device may include two separatesyringes, one may be filled with the self-assembling polypeptide and onemay be filled with the factor enhancing self-assembly. A dual syringetwo component application combination device may also be used.

It is further preferred that

-   -   i) the self-assembling polypeptide is comprised in a        composition, and/or    -   ii) the factor enhancing self-assembly is comprised in a        composition.

The composition may be a solid composition such as a powderycomposition, a liquid composition such as an aqueous composition, e.g.an aqueous solution, an emulsion, or a suspension, an aerosol such as adry or liquid aerosol, a pump spray, or a pasty composition. Preferably,the composition comprising the self-assembling polypeptide and thecomposition comprising the factor enhancing self-assembly are of thesame type. More preferably, the self-assembling polypeptide is comprisedin a liquid composition, particularly in an aqueous composition, e.g. anaqueous solution, and/or the factor enhancing self-assembly is comprisedin a liquid composition, particularly aqueous composition, e.g. aqueoussolution.

As to the definition of the term “pharmaceutical compound” and as topreferred embodiments of the “pharmaceutical compound”, “tissue”,“skin”, “mucosa”, and “hair”, it is referred to the fourth aspect of thepresent invention.

The eighth aspect of the present invention, as described above, canalternatively be worded as follows: In an eighth aspect, the presentinvention relates to a method of using an application combinationcomprising a self-assembling polypeptide and a factor enhancingself-assembly to glue one or more pharmaceutical compounds on tissue,skin, mucosa, and/or hair.

The self-assembling polypeptide being part of the applicationcombinations described with respect to the fifth to eight aspect of thepresent invention may be comprised in a composition such as an aqueoussolution, and/or the factor enhancing self-assembly being part of theapplication combinations described with respect to the fifth to eightaspect of the present invention may be comprised in a composition suchas an aqueous solution having a temperature of 20° C. and a pH ofbetween 4.5 and 7.0 such as of between 4.5 and 6.5, between 4.8 and 5.9,or of between 5.4 and 5.9. The application combinations comprising theself-assembling polypeptide and the factor enhancing self-assembly maybe applied to tissue, skin, mucosa, and/or hair. The adhesive effect ofthe self-assembling polypeptide and/or the factor enhancingself-assembly can be improved by increasing the temperature of thecomposition such as aqueous solution above 20° C., e.g. from atemperature of 20° C. to 25° C., 30° C., 35° C., 37° C., 38° C., 39° C.or 40° C. Alternatively or additionally, the adhesive effect of theself-assembling polypeptide and/or factor enhancing self-assembly can beimproved by decreasing the pH of the composition such as aqueoussolution by 0.1 to 2.5 pH units, e.g. by 0.1 to 0.5 pH units, below thepH of the tissue, skin, mucosa, and/or hair to be treated. The tissue,skin, mucosa, and/or hair to be treated may have a pH of between 4.5 and7.0 such as of between 4.5 and 6.5, of between 4.8 and 5.9, or ofbetween 5.4 and 5.9.

In a ninth aspect, the present invention relates to a self-assemblingpolypeptide for use as organ protection and/or organ isolation material.As to the definition of the term “self-assembling polypeptide” and as topreferred embodiments of the “self-assembling polypeptide”, particularlysilk polypeptide, elastin, collagen, or keratin, it is referred to thefirst aspect of the present invention. For example, it is preferred thatthe self-assembling polypeptide further comprises at least one peptidewhich is capable of enhancing the adhesive effect. It is furtherpreferred that the adhesive effect is mediated via binding of thepeptide to a cell adhesion mediating protein (CAMP). It is also,additionally or alternatively, preferred that the peptide comprises atleast one CAMP recognition sequence. As to the definition of the terms“peptide”, “peptide which is capable of enhancing the adhesive effect”,“cell adhesion mediating protein (CAMP)”, and “cell adhesion” and as topreferred embodiments of the “peptide”, “peptide which is capable ofenhancing the adhesive effect”, “cell adhesion mediating protein”, “CAMPrecognition sequence”, it is referred to the first aspect of the presentinvention.

Preferably, the organ is a sense organ, preferably selected from thegroup consisting of skin, eye, ear, nose, and mouth.

The inventors of the present invention surprisingly found that theself-assembling polypeptide is suitable to protect and/or isolate anorgan, preferably a sense organ (e.g. skin or eye), particularly fromits environment, as environmental factors, for example, might influenceand/or irritate an organ (e.g. skin or eye), or area surrounding it.Examples of environmental factors include, but are not limited to,microorganisms such as bacteria, viruses and fungi (can cause infectionsor inflammations), fine dust and fine particle (comprised in the air),and foreign matter. Its suitability, for example, has been demonstratedin the safety tests (e.g. acute eye irritation study, immunogenicitytest, acute systemic toxicity test, and acute skin irritability test)described in the experimental section.

The organ may comprise an affected area, particularly an infected,inflamed or injured area, e.g. a wound. Preferably, the wound isselected from the group consisting of an abrasion, a burn, sunburn,scratch, scrap, an ulcer, a topical wound, an open wound, and a woundcaused by heat, an electric current, chemicals (e.g. acids and/orbases), a biological agent, an inflammation or an infection.

It is preferred that the self-assembling polypeptide seals the affectedarea, particularly the infected, inflamed or injured area, e.g. thewound, comprised in the organ. The affected area may be a region of theskin, where the skin is damaged or missing. It is thereby advantageousthat the self-assembling polypeptide, particularly silk polypeptide suchas spider silk polypeptide, does not form an insurmountable barrier sothat the protected and/or isolated sites can be entered and passed bycells as well as oxygen and water. For example, the cells comprised inthe affected area can migrate, the affected tissue can be rebuilt,and/or the affected tissue can be reconnected. Furthermore, theself-assembling polypeptide, particularly silk polypeptide such asspider silk polypeptide, has the advantage that it is flexible whichreduces or even abolishes the tensile force acting on the affected area,e.g. wound. In addition, the self-assembling polypeptide prevents dryingout of the isolated and/or protected organ.

In preferred embodiments, the self-assembling polypeptide covers orcoats the organ to be isolated and/or protected, for example, theaffected area, particularly infected, inflamed or injured area, e.g. thewound, comprised in the organ.

The self-assembling polypeptide may be provided in several preferentialcompositions including, but not limited to, solid compositions such aspowdery compositions, liquid compositions such as aqueous compositions,e.g. aqueous solutions, emulsions, or suspensions, aerosols such as dryor liquid aerosols, pump sprays, and pasty compositions. Thesecompositions comprising the self-assembling polypeptide may additionallycomprise one or more pharmaceutical compounds such as therapeutic ordiagnostic compounds. Thus, for example, a liquid composition (e.g.aqueous composition) comprising the self-assembling polypeptide andoptionally further comprising one or more pharmaceutical compounds isapplied to the organ to be protected and/or isolated, for example, theaffected area, particularly infected, inflamed or injured area, e.g. thewound, comprised in the organ.

The self-assembling polypeptide may further be provided as film, gel,particularly hydrogel, foam, mesh, scaffold, patch, nonwoven, or layer,particularly covering or coating layer. The film, gel, particularlyhydrogel, foam, mesh, scaffold, path, nonwoven, or layer, particularlycovering or coating layer, comprising the self-assembling polypeptidemay additionally comprise one or more pharmaceutical compounds. Thus,for example, a film, gel, particularly hydrogel, foam, mesh, scaffold,patch, nonwoven, or layer, particularly covering or coating layer,comprising the self-assembling polypeptide and optionally furthercomprising one or more pharmaceutical compounds is applied to the organto be protected and/or isolated, e.g. the affected area, particularlyinfected, inflamed or injured area, e.g. the wound, comprised in theorgan.

The self-assembling polypeptide may also be applied to severalpreferential objects including, but not limited to, meshes, scaffolds,patches, nonwovens, and implants, for example, dental implants,microchip implants, soft tissue implants such as silicone implants, orimplants with a silicone surface (e.g. cochlea implants). Due to thisapplication, the objects including, but not limited to, meshes,scaffolds, patches, implants for example, dental implants, microchipimplants, soft tissue implants such as silicone implants, or implantswith a silicone surface (e.g. cochlea implants), and nonwovens may becovered or coated, e.g. partially or completely covered or coated, withthe self-assembling polypeptide. It can be applied to these objects viadip-coating, spraying, or dropping. For dip-coating, preferentialobjects are dipped into liquid compositions such as aqueouscompositions, e.g. aqueous solutions, comprising the self-assemblingpolypeptide. Further, for spraying, liquid compositions such as aqueouscompositions, e.g. aqueous solutions, comprising the self-assemblingpolypeptide are sprayed onto preferential objects. Furthermore, fordropping, liquid compositions such as aqueous compositions, e.g. aqueoussolutions, comprising the self-assembling polypeptide are dropped ontopreferential objects. These liquid compositions comprising theself-assembling polypeptide may additionally comprise one or morepharmaceutical compounds such as therapeutic or diagnostic compounds.Thus, for example, a mesh, scaffold, patch, nonwoven, or implant coatedand/or immersed with the self-assembling polypeptide and optionallyfurther coated and/or immersed with one or more pharmaceutical compoundsis applied to the organ to be protected and/or isolated, e.g. theaffected area, particularly infected, inflamed or injured area, e.g. thewound, comprised in the organ.

The term “pharmaceutical compound”, as used herein, is defined above.Preferably, the pharmaceutical compound is selected from the groupconsisting of an anti-microbial compound, an anti-viral compound, ananti-fungal compound, an immunosuppressive compound, a growth factor, anenzyme, an anti-inflammatory compound, an anti-allergic compound, asedative compound, a protein, particularly a glycoprotein orlipoprotein, a polysaccharide, and mixtures thereof.

The ninth aspect of the present invention, as described above, canalternatively be worded as follows: In a ninth aspect, the presentinvention relates to a method of using a self-assembling polypeptide asorgan protection and/or isolation material.

In a further aspect, the present invention relates to the use of aself-assembling polypeptide as organ protection and/or isolationmaterial. As to the definition of the term “self-assembling polypeptide”and as to preferred embodiments of the “self-assembling polypeptide”,particularly silk polypeptide, elastin, collagen, or keratin, it isreferred to the first aspect of the present invention. For example, itis preferred that the self-assembling polypeptide further comprises atleast one peptide which is capable of enhancing the adhesive effect. Itis further preferred that the adhesive effect is mediated via binding ofthe peptide to a cell adhesion mediating protein (CAMP). It is also,additionally or alternatively, preferred that the peptide comprises atleast one CAMP recognition sequence. As to the definition of the terms“peptide”, “peptide which is capable of enhancing the adhesive effect”,“cell adhesion mediating protein (CAMP)”, and “cell adhesion” and as topreferred embodiments of the “peptide”, “peptide which is capable ofenhancing the adhesive effect”, “cell adhesion mediating protein”, “CAMPrecognition sequence”, it is referred to the first aspect of the presentinvention.

Preferably, the organ is a sense organ, preferably selected from thegroup consisting of skin, eye, ear, nose, and mouth. The organ maycomprise an affected area, particularly an infected, inflamed or injuredarea, e.g. a wound. Preferably, the wound is selected from the groupconsisting of an abrasion, a burn, sunburn, scratch, scrap, an ulcer, atopical wound, an open wound, and a wound caused by heat, an electriccurrent, chemicals (e.g. acids and/or bases), a biological agent, aninflammation or an infection.

It is preferred that the self-assembling polypeptide seals the affectedarea, particularly the infected, inflamed or injured area, e.g. thewound, comprised in the organ. The affected area may be a region of theskin, where the skin is damaged or missing. In addition, theself-assembling polypeptide prevents drying out of the isolated and/orprotected organ. As to other preferred embodiments, it is referred tothe ninth aspect of the present invention.

It is another aspect of the present invention to provide a method ofconnecting two tissues by an adhesive comprising the steps of:

-   -   i) applying a self-assembling polypeptide to a first tissue, and    -   ii) contacting the first tissue with a second tissue, thereby        connecting said two tissues.

Said tissues may particularly be tissue layers. The self-assemblingpolypeptide as tissue adhesive may be applied as part of a compositionsuch as an aqueous solution having a temperature of 20° C. and a pH ofbetween 4.5 and 7.0 such as of between 4.5 and 6.5, between 4.8 and 5.9,or of between 5.4 and 5.9 to the first tissue. The adhesive effect ofthe self-assembling polypeptide can be improved by increasing thetemperature of the composition such as aqueous solution above 20° C.,e.g. from a temperature of 20° C. to 25° C., 30° C., 35° C., 37° C., 38°C., 39° C. or 40° C. Alternatively or additionally, the adhesive effectof the self-assembling polypeptide can be improved by decreasing the pHof the composition such as aqueous solution by 0.1 to 2.5 pH units, e.g.by 0.1 to 0.5 pH units, below the pH of the tissue to be connected. Thetissue to be connected may have a pH of between 4.5 and 7.0 such as ofbetween 4.5 and 6.5, of between 4.8 and 5.9, or of between 5.4 and 5.9.

It is preferred that the method further comprises the step of applying afactor enhancing self-assembly to the first tissue. This step ispreferably carried out subsequent to step i). The self-assemblingpolypeptide and the factor enhancing self-assembly may also be appliedat the same time, e.g. comprised in a two component applicationcombination device or in form of a two component application combination(see above), in step i). The self-assembling polypeptide may becomprised in a composition and/or the factor enhancing self-assembly maybe comprised in a composition. The composition may be a solidcomposition such as a powdery composition, a liquid composition such asan aqueous composition, e.g. an aqueous solution, an emulsion, or asuspension, an aerosol such as a dry or liquid aerosol, a pump spray,and a pasty composition. Preferably, the composition comprising theself-assembling polypeptide and the composition comprising the factorenhancing self-assembly are of the same type. The composition comprisingthe self-assembling polypeptide and/or the composition comprising thefactor enhancing self-assembly may comprise one or more pharmaceuticalcompounds. The term “pharmaceutical compound”, as used herein, isdefined above. Preferably, the pharmaceutical compound is selected fromthe group consisting of an anti-microbial compound, an anti-viralcompound, an anti-fungal compound, an immunosuppressive compound, agrowth factor, an enzyme, an anti-inflammatory compound, ananti-allergic compound, a sedative compound, a protein, particularly aglycoprotein or lipoprotein, a polysaccharide, and mixtures thereof. Theself-assembling polypeptide may be dried before or after the contactwith the tissue.

All aspects of the first, second, fifth, and sixth aspect of theinvention also apply to this aspect of the invention.

It is also another aspect of the present invention to provide a methodof filling a gap in a tissue by an adhesive comprising the steps of:

-   -   i) providing a tissue with a gap, and    -   ii) applying a self-assembling polypeptide to the gap in the        tissue, thereby filling the gap in the tissue.

The self-assembling polypeptide as tissue adhesive may be applied aspart of a composition such as an aqueous solution having a temperatureof 20° C. and a pH of between 4.5 and 7.0 such as of between 4.5 and6.5, between 4.8 and 5.9, or of between 5.4 and 5.9 to the gap in thetissue. The adhesive effect of the self-assembling polypeptide can beimproved by increasing the temperature of the composition such asaqueous solution above 20° C., e.g. from a temperature of 20° C. to 25°C., 30° C., 35° C., 37° C., 38° C., 39° C. or 40° C. Alternatively oradditionally, the adhesive effect of the self-assembling polypeptide canbe improved by decreasing the pH of the composition such as aqueoussolution by 0.1 to 2.5 pH units, e.g. by 0.1 to 0.5 pH units, below thepH of the tissue to be treated. The tissue to be treated may have a pHof between 4.5 and 7.0 such as of between 4.5 and 6.5, of between 4.8and 5.9, or of between 5.4 and 5.9.

It is preferred that the method further comprises the step of applying afactor enhancing self-assembly to the gap in the tissue. This step ispreferably carried out subsequent to step ii). The self-assemblingpolypeptide and the factor enhancing self-assembly may also be appliedat the same time, e.g. comprised in a two component applicationcombination device or in form of a two component application combination(see above), in step ii). The self-assembling polypeptide may becomprised in a composition and/or the factor enhancing self-assembly maybe comprised in a composition. The composition may be a solidcomposition such as a powdery composition, a liquid composition such asan aqueous composition, e.g. an aqueous solution, an emulsion, or asuspension, an aerosol such as a dry or liquid aerosol, a pump spray,and a pasty composition. Preferably, the composition comprising theself-assembling polypeptide and the composition comprising the factorenhancing self-assembly are of the same type. The composition comprisingthe self-assembling polypeptide and/or the composition comprising thefactor enhancing self-assembly may comprise one or more pharmaceuticalcompounds. The term “pharmaceutical compound”, as used herein, isdefined above. Preferably, the pharmaceutical compound is selected fromthe group consisting of an anti-microbial compound, an anti-viralcompound, an anti-fungal compound, an immunosuppressive compound, agrowth factor, an enzyme, an anti-inflammatory compound, ananti-allergic compound, a sedative compound, a protein, particularly aglycoprotein or lipoprotein, a polysaccharide, and mixtures thereof. Theself-assembling polypeptide may be dried before or after the contactwith the tissue gap.

All aspects of the first, second, fifth, and sixth aspect of theinvention also apply to this aspect of the invention.

In a further aspect, the present invention relates to a spider silkpolypeptide characterized by a size of no more than 100 kDa, preferablyof no more than 95 or 90 kDa, more preferably of no more than 85 or 80kDa, even more preferably of no more than 75 or 70, most preferably ofno more than 65 kDa, comprising at least one, preferably one or two,non-repetitive sequence(s) according to SEQ ID NO: 76 (NR5) or SEQ IDNO: 77 (NR6). Preferably, the spider silk polypeptide is a recombinantspider silk polypeptide. It is also preferred that the spider silkpolypeptide is C₁₆NR5 or C₁₆NR6.

In another further aspect, the present invention relates to a spidersilk polypeptide consisting of no more than 1000 amino acids, preferablyof no more than 950 amino acids, more preferably of no more 900 aminoacids, even more preferably of no more than 850 amino acids, mostpreferably of no more than 800 or 750 amino acids, comprising at leastone, preferably one or two, non-repetitive sequence(s) according to SEQID NO: 76 (NR5) or SEQ ID NO: 77 (NR6). Preferably, the spider silkpolypeptide is a recombinant spider silk polypeptide. It is alsopreferred that the spider silk polypeptide is C₁₆NR5 or C₁₆NR6.

In a further aspect, the present invention relates to an implant coatedwith a self-assembling polypeptide. As to the definition of the term“self-assembling polypeptide” and as to the preferred embodiments of the“self-assembling polypeptide”, it is referred to the first aspect of thepresent invention. In particular, the self-assembling polypeptide isselected from the group consisting of a silk polypeptide such as arecombinant silk polypeptide. Preferably, the implant is a dentalimplant, a microchip implant, or a soft tissue implant, such as asilicone implant, or an implant with a silicone surface (e.g. a cochleaimplant). The silicone implant or implant with a silicone surface mayalso be a breast implant. The term “coating” in this respect, refers toa covering that is comprised on the implant. The implant may bepartially or completely covered or coated with the self-assemblingpolypeptide. Preferably, said “coating” completely covers or surroundsthe implant. It is preferred that the “coating” has a thickness ofbetween 1 nm and 50 μm, preferably 40 nm and 50 μm, more preferablybetween 0.5 μm and 10 μm and most preferably between 0.5 μm and 5 μm.

In a further aspect, the present invention relates to a self-assemblingpolypeptide for use as a coating material or to the use of aself-assembling polypeptide as a coating material. As to the definitionof the term “self-assembling polypeptide” and as to the preferredembodiments of the “self-assembling polypeptide”, it is referred to thefirst aspect of the present invention. In particular, theself-assembling polypeptide is selected from the group consisting of asilk polypeptide such as a recombinant silk polypeptide.

In another further aspect, the present invention relates to acomposition comprising a self-assembling polypeptide and a factorenhancing self-assembly for use as a coating material or to the use of acomposition comprising a self-assembling polypeptide and a factorenhancing self-assembly as a coating material. Preferably, the factorenhancing self-assembly is selected from the group consisting ofalcohols, sulfates, phosphates, and a cross-linking agent.

The term “cross-linking” agent refers to a compound which is able toform chemical links between molecular chains such as protein chains tobuild a three-dimensional network of connected molecules such asproteins. As to the preferred embodiments of the “cross-linking agent”,it is referred to the fifth aspect of the present invention. It isparticularly preferred that the cross-linking agent is genipin.

Said composition includes, but is not limited to, a solid compositionsuch as a powdery composition, a liquid composition such as an aqueouscomposition, e.g. an aqueous solution, emulsion, or suspension, anaerosol such as a dry or liquid aerosol, a pump spray, and a pastycomposition. The composition comprising the self-assembling polypeptideand the factor enhancing self-assembly may additionally comprise one ormore pharmaceutical compounds such as therapeutic or diagnosticcompounds. Thus, for example, a liquid composition (e.g. aqueouscomposition) comprising the self-assembling polypeptide, the factorenhancing self-assembly and one or more pharmaceutical compounds such astherapeutic or diagnostic compounds may be used as a coating material.

Various modifications and variations of the invention will be apparentto those skilled in the art without departing from the scope ofinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled in the artin the relevant fields are intended to be covered by the presentinvention.

The following figures and examples are merely illustrative of thepresent invention and should not be construed to limit the scope of theinvention as indicated by the appended claims in any way.

FIGURE LEGEND

FIG. 1: Production of C₁₆spRGD and ntag^(Cys)C₁₆-c(RGDfK). (A) Chemicalstructure of the synthetic cyclic RGD peptide c(RGDfK)-spacermoiety-part of SMCC employed for chemical modification of ntag^(Cys)C₁₆.(B) eADF4 (C₁₆), the RGD-containing variant ntag^(Cys)C₁₆-c(RGDfK)(chemically modified) and C₁₆spRGD (genetically modified). Forntag^(Cys)C₁₆-c(RGDfK), c(RGDfK)-spacer moiety-part of SMCC wascovalently coupled to the thiol-group of a cysteine residue ofntag^(Cys)C₁₆. C₁₆spRGD was modified by genetic engineering hybridizinga spacer and an RGD domain with eADF4 (C₁₆).

FIG. 2: Preparation of the pig skin and application of the C₁₆ orC₁₆spRGD solution to the pig skin for the adhesive test. (A) Pig skinwas used for the adhesive test. The pig skin was cut into pieces havinga size of about 9.5×11.0 cm (a size which fits in a Petri dish having adiameter of 14.5 cm) using a scalpel. (B) The pieces were fixed at theiredges with nails onto a board to tighten the skin. (C) and (D) Threeincisions per piece of skin were made according to an exemplary sampleof 1 cm×1 cm using a scalpel. (E) The resulting skin graft wassubsequently lifted with tweezers and cut to produce a skin pocket. (F)The C₁₆ or C₁₆spRGD solution was dropped on the cutting area using apipette (at low protein concentrations of <40 mg/ml) or spread on thecutting area using a spatula (at high protein concentrations of ≥40mg/ml) to cover the surface of the “wound”. (G) The separated skin flapwas subsequently repositioned on the cutting area.

FIG. 3: Adhesive test. The adhesive effect of C₁₆ or C₁₆spRGD was testedby bending (e.g. rolling) the pig skin piece as prepared above (see FIG.2). The bending (e.g. rolling) was carried out in order to apply tensionto the “wound”. (A) Photographic picture of a bended pig skin piececomprising a cutting area treated with a C₁₆ solution. The skin flapremained connected to the treated cutting area. (B) Schematic picture ofthe bended pig skin piece as shown in (A). (C) Shows a skin flap beforethe application of a C₁₆spRGD solution and (D) Shows a skin flap afterapplication of a C₁₆spRGD solution (concentration: 40 mg/ml andincubation time: 1 hour). The dotted line represents the cutting edgesof the skin flap.

FIG. 4: Preparation of the pig skin and application of the C₁₆ orC₁₆spRGD solution to the pig skin for the pulling test. (A) Pig skin wasused for the pulling test. Pig skin stripes having a size of 1.5 cm×6 cmwere generated. One vertical incision of 1.5 cm in length was made at adistance of 2.5 cm from the short end of the stripe using a scalpel (seecontinuous line within the pig skin stripe). (B) The incision wasterminated at half of the thickness (the thickness is marked with a “d”)of the pig skin piece (see incision marked with “a”). The incision wascontinued horizontally for 1 cm (see incision marked with “b”) beforeturning in vertical direction to cut the tissue (see incision markedwith “c”). The incision to cut the tissue is also indicated as dottedline within the pig skin stripe in (A). (C) The cutting areas/surfacesof the produced two pig skin strip halves were coated with a C₁₆ orC₁₆spRGD solution.

FIG. 5: (A) a super glue (e.g. “UHU Kunststoff Spezialsekundenkleber”)was applied to the skin side of the stripe. The super glue was spreadusing a spatula or cell scraper. No glue was applied in near vicinity ofthe cutting site. Plastic slides (e.g. “Rinzle plastic micro-slides”)having a length of 2.5 and 3.5 cm were subsequently connected with theglued site of the skin stripe (the slide having a length of 2.5 cm waspositioned on the short site of the skin slide and the slide having alength of 3.5 cm was positioned on the longer site of the skin slide).(B) The same was done for the side opposite to the skin side of thestripe. (C) The adhesion force under tangential stress was tested usinga tensile tester. Therefore, the glued pig skin stripe was fixed in thetensile tester using sample holders. The arrows indicate the directionof movement.

FIG. 6: Visual analysis of silk protein coated or uncoated implantsafter submuscular implantation in the back of rats. (A) and (B) Theuncoated implants showed capsular fibrosis. (C) The capsular fibrosis inthe silk protein coated implants was strongly reduced (the capsule wasthinner). In addition, no scarring was visible.

FIG. 7: Comparison of the capsule thickness: implants coated with thesilk adhesive and non-coated implants (control).

FIG. 8: Immunogenicity test. Endpoint IgG titers 2, 5 and 8 weeks afteradministration of eADF4 (C₁₆). The entire group of five mice showed noor no significant increase of IgG and therefore no or no significantspecific antibody formation.

Examples

1. Production of eADF4 (C₁₆), C₁₆spRGD, and ntagCysC₁₆-c(RGDfK)1.1 Production of eADF4 (C₁₆)The recombinant spider silk protein eADF4 (also designated as C₁₆herein) is based on the consensus sequence of one of three spidroins ofthe dragline silk of the European garden spider (Araneus diadematus).The consensus motif (C module) of ADF4(GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 21)) is repeated 16times in the recombinant protein (FIG. 1B). For detection, an N-terminalT7-tag may be attached to the molecule. Production in E. coli andpurification was performed as described in WO 2011/120690 A2(“Separation of insoluble target proteins”).1.2 Genetic modification of eADF4 (C₁₆)DNA cassettes encoding RGD and a spacer sequence were created byannealing two synthetic oligonucleotides. For the RGD-tag:GATCCATGGGCGGTCGTGGTG ACTCTCCGGGTTAATGAA (SEQ ID NO: 72) andAGCTTTCATTAACCCGGAGAGTCACCACGACCGCCCATG (SEQ ID NO: 73) and for thespacer sequence: GATCCATGGGCGGTGGCTCTGGTTAATGAA (SEQ ID NO: 74) andAGCTTT CATTAACCAGAGCCACCGCCCATG (SEQ ID NO: 75) were used. The resultingamino acid sequence for the specific tag spRGD was GGSGGRGDSPG (SEQ IDNO: 53) (FIG. 1B). The insertion of the DNA sequences into the cloningvector and the ligation with the gene encoding eADF4 (C₁₆) wereaccomplished by a seamless cloning strategy as described previously byHuemmerich et al. (“Primary structure elements of spider dragline silksand their contribution to protein solubility”, Biochemistry, 2004, 43:13604-12). The DNA sequence of the genetically engineered C₁₆spRGD wasconfirmed by sequencing. Protein production and purification procedureswere identical to that of eADF4 (C₁₆) (see above). A sequence of thegenetically engineered C₁₆spRGD including a T7 Tag is shown in SEQ IDNO: 78.1.3 Chemical coupling of RGD to a cysteine-modified variant of eADF4(C₁₆)For high coupling specificity, chemical coupling of RGD peptides wasperformed with the cysteine containing eADF4 (C₁₆) variant ntag^(Cys)C₁₆which has been previously established by Spiess et al. (“Structuralcharacterization and functionalization of engineered spider silk films”,Soft Matter, 2010, 6: 4168-74) (FIG. 1B) (ntag^(Cys) also designated asTAG^(CYS3) herein, has a sequence according to SEQ ID NO: 37, and C₁₆comprises 16 times module C having a sequence according to SEQ ID NO:21). For coupling of the cyclic RGD c(RGDfK)-spacer moiety-part of SMCC(SMCC=Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (seePeptides International, Louisville, Ky., USA) (see also Pierschbacherand Ruoslahti, “Influence of stereochemistry of the sequenceArg-Gly-Asp-Xaa on binding specificity in cell adhesion”, J Biol Chem,1987, 262: 17294-8; Haubner et al. “Stereoisomeric peptide libraries andpeptidomimetics for designing selective inhibitors of thealpha(V)beta(3) integrin for a new cancer therapy”, Angew. Chem. Int.Edit., 1997, 36: 1375-89; and Aumailley et al. “Arg-Gly-Asp constrainedwithin cyclic pentapeptides, Strong and selective inhibitors of celladhesion to vitronectin and laminin fragment P1”, FEBS Lett, 1991, 291:50-4) (FIG. 1A), lyophilized ntag^(Cys)C₁₆ was dissolved in 6 Mguanidinium thiocyanate (GdmSCN), dialyzed against 20 mM HEPES, pH 7,and diluted to a final concentration of 2 mg/ml. For reduction ofdisulfide bonds, proteins were incubated in a ten-fold excess oftris(2-carboxyethyl)phosphine (TCEP) for 2 h at RT. After addition of atwenty-fold excess of c(RGDfK)-spacer moiety-part of SMCC, the reactionof maleimide and free thiol-groups was carried out for 2 h at RT. Theprotein was purified by precipitation with potassium phosphate (pH 8) ata final concentration of 1 M, followed by washing the pellet three timeswith deionized water.2. Preparation of the silk protein solutionC₁₆ and C₁₆spRGD have been produced as described above. Respectiveamounts of C₁₆ and C₁₆spRGD were dissolved in 6M Guanidinium thiocyanateand dialyzed for at least 3 days at 4° C. against 5 mM Tris, pH 9. Afterdialysis, the samples were centrifuged for 15 min at 14.000 rpm at 4° C.Then the protein concentration was estimated by UV-Vis spectrometer. Theprotein solutions were diluted with 5 mM Tris solution, pH 9 to therequested concentrations.3. Adhesive test3.1 Preparation of skinPig skin was used for the adhesive test. The pig skin was cut intopieces having a size of about 9.5×11.0 cm (a size which fits in a Petridish having a diameter of 14.5 cm) using a scalpel. The pieces werefixed at their edges with nails onto a board to tighten the skin (seeFIGS. 2A and B). Three incisions per piece of skin were made accordingto an exemplary sample of 1 cm×1 cm using a scalpel (see FIGS. 2C andD). The resulting skin graft was subsequently lifted with tweezers andcut to produce a skin pocket as shown in FIG. 2E.3.2 Application of the silk protein solution to the skinThe C₁₆ or C₁₆spRGD solution as produced above was dropped on thecutting area using a pipette (at low protein concentrations of <40mg/ml), or spread on the cutting area using a spatula or cell scraper(at high protein concentrations of ≥40 mg/ml) to cover the surface ofthe “wound” (see FIG. 2F). The separated skin flap was subsequentlyrepositioned on the cutting area (see FIG. 2G). The cutting area mayalso be designated as cutting surface. The samples were incubated at 37°C.

The adhesive effect was tested by bending (e.g. rolling) the pig skinpiece as prepared above (see FIGS. 3A and 3B). The bending (e.g.rolling) was carried out in order to apply tension to the “wound”. Ifthe skin flap remained connected to the cutting area treated with theC₁₆ or C₁₆spRGD solution during bending (e.g. rolling), the sample wasgraded as a sample showing adhesive properties. If the skin flapdetached from the cutting area treated with the C₁₆ or C₁₆spRGD solutionduring bending (e.g. rolling), the sample was graded as a sample showingno adhesive properties.

In the following, the results of the adhesive test using pig skin piecestreated with C₁₆spRGD solutions having a concentration of 30, 40, 50,and 70 mg/ml (see Tables 2 and 3), or C₁₆ solutions having aconcentration of 40, 50, and 70 mg/ml (see Table 3) are shown.

TABLE 2 Results of a first C₁₆spRGD adhesive test with different silkprotein concentrations at an incubation time of 15 min and 1 h C₁₆spRGDconcentration [mg/ml] after 15 min after 1 h 30

✓ 40 + ✓

: no adhesive effect, +: adhesive effect at the edges of the cuttingsurface, ✓: adhesive effect at the complete cutting surface

TABLE 3 Results of a second C₁₆ and C₁₆spRGD adhesive test withdifferent silk protein concentrations and volumes at an incubation timeof 1 h Concentration Volume [μl/cm²] [mg/ml]  C₁₆ C₁₆spRGD References 5070 ✓ ✓ Ref-Tris

50 ✓ ✓ Ref 40 + +

25 70 ✓ ✓ Ref-Tris

50 ✓ ✓ Ref 40 ✓ ✓

Ref-Tris: Tris buffer Ref: H₂O

: no adhesive effect, +: adhesive effect at the edges of the cuttingsurface, ✓: adhesive effect at the complete cutting surfaceAn adhesive effect of C₁₆ and C₁₆spRGD has been shown for all samplestreated with C₁₆ and C₁₆spRGD solutions (concentration 40 to 70 mg/ml,volume of 50 μl/cm² and concentration 40 to 70 mg/ml, volume of 25μl/cm²) after an incubation time of 1 hour. In each case, the skin flapremained connected to the cutting area during bending (see, for example,FIGS. 3A and B for C₁₆). No adhesive effect has been shown for bothreference samples (Ref-Tris: cutting area treated with Tris buffer andRef: cutting area treated with H₂O) used as negative controls. In thesesamples, the skin flap detached from the cutting area during bending.FIG. 3C further shows a skin flap before the application of the C₁₆spRGDsolution and FIG. 3D shows a skin flap after application of the C₁₆spRGDsolution (concentration: 40 mg/ml and incubation time: 1 hour). Thedotted line represents the cutting edges of the skin flap.

Similar results were achieved with pig skin pieces having the skin flapcompletely removed. The cutting area was covered with the C₁₆ andC₁₆spRGD solutions as described above. Afterwards, the skin flap wasrepositioned on the cutting area. The adhesive test was carried out asdescribed above.

4. Pulling Test 4.1 Preparation of Skin

Pig skin was used for the pulling test. The pig skin was cut into pieceshaving a size of about 6.0×11.0 cm using a scalpel. The pieces werefixed at their edges with nails onto a board to tighten the skin. In anext step, pig skin stripes having a size of 1.5 cm×6 cm were cut outfrom the pig skin pieces (see FIG. 4A). The single pig skin stripes werefurther processed as follows: One vertical incision of 1.5 cm in lengthwas made at a distance of 2.5 cm from the short end of a stripe using ascalpel (see continuous line within the pig skin stripe in FIG. 4A). Theincision was terminated at half of the thickness (the thickness ismarked with a “d” in FIG. 4B) of the pig skin stripe (see incisionmarked with “a” in FIG. 4B). The incision was continued horizontally for1 cm (see incision marked with “b” in FIG. 4B) before turning invertical direction to cut the tissue (see incision marked with “c” inFIG. 4B and dotted line within the pig skin stripe in FIG. 4A).4.2 Application of the silk protein solution to the skinThe above described incision/cutting procedure separated the pig skinstripe in two halves. The pig skin stripe halves were separated fromeach other to treat the cutting areas with the C₁₆ or C₁₆spRGD solutionas produced above. Therefore, the C₁₆ or C₁₆spRGD solution was droppedon the cutting areas using a pipette (at low protein concentrations of<40 mg/ml), or spread on the cutting areas using a spatula or cellscraper (at high protein concentrations of ≥40 mg/ml) to cover thesurface of the “wound”. The two pig skin stripe halves were subsequentlyrepositioned so that the treated cutting areas came in contact with eachother (see FIG. 4C). The samples were incubated at 37° C. for 30 min.4.3 Sample preparation for pulling testUpon expiry of the incubation time of 30 min, a super glue (e.g. “UHUKunststoff Spezialsekundenkleber”) was applied to the skin side of thestripe. The super glue was spread using a spatula or cell scraper. Noglue was applied to the cutting site. Plastic slides (e.g. “Rinzleplastic micro-slides”) having a length of 2.5 and 3.5 cm weresubsequently connected with the glued site of the skin stripe (the slidehaving a length of 2.5 cm was positioned on the short site of the skinslide and the slide having a length of 3.5 cm was positioned on thelonger site of the skin slide) (see FIG. 5A). The same was done for theside opposite to the skin side of the stripe (see FIG. 5B).The adhesion force under tangential stress was tested using a tensiletester (e.g. Zwicki Z 0.5; Zwick Roell, 50 N load cell). Therefore, theglued pig skin stripe was fixed in the tensile tester using sampleholders as shown in FIG. 5C.The parameters of the pulling test were as follows:

Preload: 0.01 MPa

Testing speed: 10 mm/minClamping length at starting position: 15.00 mmSpeed tensile modulus: 10 mm/minIn the following, the results of the pulling test using pig skin stripestreated with C₁₆spRGD or C₁₆ solutions having a concentration of 35mg/ml are shown.

TABLE 4 Results of the C₁₆ and C₁₆spRGD pulling test Concentration [35mg/ml] of Incubation time Average σ_(max) (N) C₁₆ 30 min 2.92 C₁₆spRGD30 min 5.29The average maximal adhesion strength is indicated in Table 4. Themaximal adhesion strength can be defined as the maximal load per unitwidth of the bond line required to produce progressive separation of twobonded adherents, particularly flexible adherents. The average maximaladhesion strength for C₁₆spRGD was increased by about 80% compared tothe average maximal adhesion strength for C₁₆.

The above experimental data clearly demonstrate that silk proteins (e.g.C₁₆) or modified silk proteins (e.g. C₁₆spRGD) function as tissueadhesives. Thus, silk proteins can be used, for example, as tissueadhesives to treat wounds or sutured wounds.

5. Implant coating with silk proteinsTextured silicone implants (Polytech Health & Aesthetics/Germany) havinga diameter of 2.6 cm and a volume of 3 ml were covered with a silkprotein layer of a thickness of 10 μm according to the followingprotocol: C₁₆ protein was produced as described above. 1.35 g of C₁₆protein was dissolved in 135 ml of 6M Guanidinium Thiocyanate undergentle agitation. 135 ml of 50 mM Tris buffer (pH 9 (Roth) 4° C.) wasslowly added to obtain a homogeneous solution. The resulting proteinsolution was dialyzed overnight against 50 mM Tris buffer pH 9 at 4° C.Guanidinium-SCN remnants were removed via cross-flow filtration at 4° C.while Tris buffer (50 mM, pH 9) was constantly added. Subsequently theC₁₆ protein solution was concentrated to 60 ml. The final concentrationwas 10.8 mg/ml (determined by UV/Vis-Spectroscopy, Beckman Coulter, DU800).

The coating was performed in a sterile chamber (sterilized at 140° C.for 1 hour). The silicone implants were washed with ethanol and dried atRT prior to the coating process. The silicone implants were coated 3times with the C₁₆ protein solution (30 ml at 10.8 mg/ml) by dipping thesilicone implants into the solution for 120 s and drying at air for 300s, respectively. For post-treatment, the transplants were dipped inKH₂PO₄ solution (1M pH4 (Roth, 99%), NaCl 0.91% w/v (Roth, 99.5%)) for120 s and dried for 120 s before washing the transplants in a salinesolution (9 g/l). After sterilization by gamma-irradiation with a doseof 5 kGray (at Isotron, Allershausen), the silk protein coated implantswere implanted submuscular in the back of Sprague-Dawley rats having aweight of 250 to 300 mg. As a control, uncoated implants were used.

After 3 months, the Sprague-Dawley rats were sacrificed. The implantswere exposed and subsequently analyzed. The results are illustrated inFIG. 6. While the uncoated implants showed capsular fibrosis (see FIGS.6A and B), the capsular fibrosis in the silk protein coated implants wasstrongly reduced (see FIG. 6C). This results in less scarring tissuebeing formed at the interface of the implant and the host tissue, asevident by a 30% reduction of the scarring tissue forming the implantcapsule compared to the control group (see FIG. 7). Further, thecapsules of the silk protein coated implants were thinner (see FIG. 6C)in contrast to the capsules of the uncoated implants (see FIG. 6A).These data allow the conclusion that wounds which are glued withself-assembling proteins, particularly silk proteins, exhibit reduced orno scarring and/or reduced or no fibrosis, particularly capsularfibrosis.6. Safety tests6.1 Acute Eye Irritation testThe acute eye irritation test was performed according to DIN EN ISO1093-1 und GLP conditions. Particularly, 0.3 mg spider silk proteineADF4 (C₁₆) dissolved in 100 μl phosphate-buffered saline was applied toone of the two eyes of three female New Zealand white rabbits. Thenon-treated eye of each female New Zealand white rabbit was taken as acontrol. This treatment did not cause any signs of pain and did notresult in any clinical findings. It showed neither eye damage (a risk ofserious damage to the eyes could be excluded according to GHS H 318(Global Harmonizing System)) nor eye irritation (eADF4 (C₁₆) was notclassified as irritant according to GHS H 319).6.2 Immunogenicity testA composition of eADF4 (C₁₆) was administered subcutaneously to fivefemale BALB/c mice at final doses of 2, 10 and 50 μg, respectively. Oneweek before and two, five and eight weeks after administration, serawere harvested and analyzed for the presence of antibodies directedagainst the test substances. The entire group of five mice showed nosignificant specific antibody formation (see FIG. 8).6.3 Acute Systemic Toxicity testThe Acute systemic toxicity test was performed according to DIN EN ISO10993-11 under GLP conditions. Particularly, eADF4 (C₁₆) was given oncei.p. at a dose of 250 mg/kg to female NMRI mice. Two groups of fivefemale mice each were tested, one with the test item dissolved inphosphate-buffered saline, one with the vehicle. No test item groupanimal showed any clinical findings at the end of the observationperiod. There was no significant change of body weight. No furtherfindings, such as macroscopic findings or change of organ weights werenoted during the observation period.6.4 Acute Skin Irritability testThe acute skin irritability test was performed according to OECD 404guidelines und GLP conditions. Particularly, an ADF4 (C₁₆) film patch of42.5 mg and 6 cm² area was moistened with 100 μl saline, applied topreviously shaved skin on the backs of each of three male New Zealandwhite rabbits, and fixated with sterile gauze pads and hypoallergicplaster. After four hours incubation the film patches were removed andthe treated skin was examined. The treatment with a eADF4 (C₁₆) film didnot cause any erythema formation or edema formation directly after theapplication or during the observation period. No general clinicalfindings and no initial pain reaction were observed afteradministration.

1.-55. (canceled)
 56. A method for adhering tissue comprising the steps of: (i) providing a self-assembling polypeptide, (ii) applying the self-assembling polypeptide to at least one of at least two tissue layers, thereby coating the at least one of the at least two tissue layers with the self-assembling polypeptide, and (iii) contacting the at least two tissue layers, thereby adhering the at least two tissue layers together.
 57. The method of claim 56, wherein said polypeptide further comprises at least one peptide which is capable of enhancing the adhesive effect.
 58. The method of claim 57, wherein the adhesive effect is mediated via binding of the peptide to a cell adhesion mediating protein (CAMP).
 59. The method of claim 58, wherein the peptide comprises at least one CAMP recognition sequence.
 60. The method of claim 59, wherein the CAMP recognition sequence comprises a module containing RGD, GER, GEK, GEN, IDAPS (SEQ ID NO: 45) or variants thereof, GPR, HHLGGAKQAGDV (SEQ ID NO: 46) or variants thereof, CDPGYIGSR (SEQ ID NO: 47) or variants thereof, AEIDGIEL (SEQ ID NO: 48) or variants thereof, QIDS (SEQ ID NO: 49), or LTD.
 61. The method of claim 56, wherein the self-assembling polypeptide is a silk polypeptide.
 62. The method of claim 61, wherein the silk polypeptide is selected from the group consisting of ADF-3 (SEQ ID NO: 1) or variants thereof, ADF-4 (SEQ ID NO: 2) or variants thereof, MaSp I (SEQ ID NO: 43) or variants thereof, MaSp II (SEQ ID NO: 44) or variants thereof, (C)_(m), (C)_(m)NR_(z), NR_(z)(C)_(m), (AQ)_(n), (AQ)_(n)NR_(z), NR_(z)(AQ)_(n), (QAQ)_(o), NR_(z)(QAQ)_(o), (QAQ)_(o)NR_(z), wherein m is an integer of 8 to 48, n is an integer of 6 to 24, o is an integer of 8 to 16, z is an integer of 1 to 3 and NR stands for a non-repetitive unit.
 63. The method of claim 62, wherein the silk polypeptide is C₁₆NR4, C₃₂NR4, (AQ)₁₂NR3, (AQ)₂₄NR3, (AQ)₁₂, (AQ)₂₄, C₁₆, C₃₂, NR4C₁₆NR4, NR4C₃₂ NR4, NR3C₁₆NR3, NR3C₃₂NR3, NR4(AQ)₁₂NR4, NR4(AQ)₂₄NR4, NR3(AQ)₁₂NR3, NR3(AQ)₂₄NR3, (QAQ)₈ or (QAQ)₁₆.
 64. The method of claim 58, wherein the CAMP is an integrin, a selectin, or a cadherin.
 65. The method of claim 56, wherein tissue is adhered i) in the treatment of a wound, ii) in the treatment of a sutured wound, iii) in the fixation of transplants, iv) in surgical interventions, or v) in the processing of food.
 66. The method of claim 65, wherein the wound is selected from the group consisting of a topical wound, deep wound, gaping wound, stab wound, puncture wound, penetration wound, surgical incision, laceration, cut, and trauma.
 67. The method of claim 65, wherein the surgical interventions are selected from the group consisting of cardiovascular surgical interventions, cardiothoracic surgical interventions, gastrointestinal surgical interventions, pneumothoracic surgical interventions, neurosurgical interventions, urological surgical interventions, dental surgical interventions, reconstructive surgical interventions, surgical interventions in the ear, surgical interventions in the nose, surgical interventions in the throat area, lymphatic, biliary and cerebrospinal fistulae, air leakages during thoracic and pulmonary surgical interventions, orthopaedic surgical interventions, gynaecological surgical interventions, cosmetical surgical interventions, and vascular surgical interventions.
 68. A method for connecting one or more pharmaceutical or cosmetic compounds with tissue, skin, mucosa, or hair comprising the steps of: (i) providing a self-assembling polypeptide, (iia) applying the self-assembling polypeptide to tissue, skin, mucosa, or hair, thereby coating the tissue, skin, mucosa, or hair with the self-assembling polypeptide, and (iiia) contacting one or more pharmaceutical or cosmetic compounds with the tissue, skin, mucosa, or hair coated with the self-assembling polypeptide, thereby gluing the one or more pharmaceutical or cosmetic compounds to the tissue, skin, mucosa, or hair, or (iib) applying the self-assembling polypeptide to one or more pharmaceutical or cosmetic compounds, thereby coating the one or more pharmaceutical or cosmetic compounds with the self-assembling polypeptide, and (iiib) contacting tissue, skin, mucosa, or hair with the one or more pharmaceutical or cosmetic compounds coated with the self-assembling polypeptide, thereby gluing the tissue, skin, mucosa, or hair to the one or more pharmaceutical or cosmetic compounds.
 69. The method of claim 68, wherein the cosmetic compounds are selected from the group consisting of dyes, fragrances, stem cells, enzymes, vitamins, UV protective agents, and antioxidants, or the pharmaceutical compounds are selected from the group consisting of anti-microbial compounds, anti-viral compounds, anti-fungal compounds, immunosuppressive compounds, growth factors, enzymes, anti-inflammatory compounds, anti-allergic compounds, sedative compounds, proteins, polysaccharides, and mixtures thereof.
 70. The method of claim 68, wherein the self-assembling polypeptide is a silk polypeptide.
 71. A method for connecting a tissue or skin layer and an artificial object comprising the steps of: (i) providing a self-assembling polypeptide, (iia) applying the self-assembling polypeptide to at least one tissue or skin layer, thereby coating the at least one tissue or skin layer with the self-assembling polypeptide, and (iiia) contacting at least one surface of an artificial object with the at least one tissue or skin layer coated with the self-assembling polypeptide, thereby connecting the at least one surface of the artificial object with the at least one tissue or skin layer, or (iib) applying the self-assembling polypeptide to at least one surface of the artificial object, thereby coating the at least one surface of the artificial object with the self-assembling polypeptide, and (iiib) contacting at least one tissue or skin layer with the at least one surface of the artificial object coated with the self-assembling polypeptide, thereby connecting the at least one tissue or skin layer with the at least one surface of the artificial object.
 72. The method of claim 71, wherein the artificial object is a medical device.
 73. The method of claim 72, wherein the medical device is selected from the group consisting of implants, microchips, cardiac pacemakers, pumps, cannula, deposits, fixations, prostheses, and sensors.
 74. The method of claim 71, wherein the self-assembling polypeptide is a silk polypeptide.
 75. A method for reducing or preventing capsular fibrosis comprising the steps of: (i) providing a self-assembling polypeptide, (ii) coating an implant with the self-assembling polypeptide, thereby reducing or preventing capsular fibrosis when implanting the implant into the human body.
 76. The method of claim 75, wherein the implant is a breast implant.
 77. The method of claim 75, wherein the self-assembling polypeptide is a silk polypeptide. 